Interplay Between the Effects of Dilated Cardiomyopathy Mutation (R206L) and the Protein Kinase C Phosphomimic (T204E) of Rat Cardiac Troponin T Are Differently Modulated by α- and β-Myosin Heavy Chain Isoforms (original) (raw)
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The FEBS Journal, 2021
We investigated the mechanisms associated with E22K mutation in myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) in humans and mice. Specifically, we characterized the mechanical profiles of papillary muscle fibers from transgenic mice expressing human ventricular RLC wild-type (Tg-WT) or E22K mutation (Tg-E22K). Because the two mouse models expressed different amounts of transgene, the B6SJL mouse line (NTg) was used as an additional control. Mechanical experiments were carried out on Ca 2+-and ATP-activated fibers and in rigor. Sinusoidal analysis was performed to elucidate the effect of E22K on tension and stiffness during activation/rigor, tension-pCa, and myosin cross-bridge (CB) kinetics. We found significant reductions in active tension (by 54%) and stiffness (active by 40% and rigor by 54%). A decrease in the Ca 2+ sensitivity of tension (by ΔpCa ~ 0.1) was observed in Tg-E22K compared with Tg-WT fibers. The apparent (=measured) rate constant of exponential process B (2πb: force generation step) was not affected by E22K, but the apparentrate constant of exponential process
Biophysical Journal, 2006
The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-D23Tm (Ala-Ser-Tm D(47-123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5-40°C for fibers activated in the presence of saturating ATP and Ca 21. The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had ;40% the control value, and the slope from mutant Tm had ;36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30-40°C); only processes B and C were identified in the mid-temperature range (15-25°C), and only process C was identified in the low temperature range (5-10°C). At a given temperature, similar apparent rate constants (2pa, 2pb, 2pc) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-D23Tm , Actin , AS-Tm % Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but D23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by D23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.
The Journal of Physiology, 1991
1. The isometric length‐tension relationship for cardiac muscle is generally steeper than for skeletal muscle in the physiological range of sarcomere lengths. Recent studies suggest that cardiac troponin C (cTnC) may have intrinsic properties that confer greater length‐dependent changes in Ca2+ sensitivity of tension than for skeletal troponin C (sTnC). We tested this hypothesis by characterizing tension‐pCa (pCa is ‐log[Ca2+]) relationships in rabbit skinned psoas muscle fibres at mean sarcomere lengths of 2.32 and 1.87 microns both before and after partial replacement of endogenous sTnC with cTnC. 2. In untreated control fibres, the mid‐point (pCa50) of the tension‐pCa relationship shifted to lower pCa by 0.15 +/‐ 0.02 pCa units, i.e. became less sensitive to Ca2+, when sarcomere length was reduced, and the relationship became steeper. 3. Partial extraction of endogenous sTnC and reconstitution with cTnC resulted in no change in the length‐dependent shift of pCa50 when reconstitut...
The Journal of Physiology, 2003
The major constituents of the contractile apparatus in striated muscle are the thick and thin filaments. Force is generated when a protrusion from the thick filament, called a cross-bridge, attaches to the thin filament and catalyses ATP hydrolysis. The thin filament is composed of actin, tropomyosin (Tm) and three subunits of the troponin (Tn) molecules. Modification of a portion of the thin filament and examination of how this modification alters its function provide an excellent method of studying the structure-function relationship. However, it has been difficult to extract Tm and to replace it with a mutant Tm, because Tm is a filamentous protein. Recently, both Ishiwata's group (Fujita et al. 1996; Fujita & Ishiwata, 1998) and our group (Fujita et al. 2002; Fujita & Kawai, 2002) have succeeded in removing the thin filament from bovine myocardium and in reconstituting the thin filament with constituent proteins. After reconstitution, sinusoidal analysis was performed to access cross-bridge kinetics. The structure of the reconstituted myocardium was examined by electron microscopy and SDS-PAGE, and its function was measured by isometric tension and cross-bridge kinetics. These methods confirmed that the reconstitution was complete both structurally and functionally. The ability to selectively remove the thin filament and to reconstitute it with component proteins offers a unique possibility to study the role of a specific domain of a thin filament protein by using genetically engineered mutant proteins. Examination of Tm mutants has been helpful in understanding the regulatory function of the thin filament. Hitchcock-DeGregori and her coworkers (Hitchcock-DeGregori & Varnell, 1990; Hitchcock-DeGregori & An, 1996) found that deletion of region 2 or 3 out of seven quasi-repeating regions does not interrupt Ca 2+ regulation. They also found that these regions contribute modestly to Tm-Tn binding to actin. Also, Landis et al. (1999) have found that it is not the length of Tm that is critical for proper regulatory function but, rather, it is the specific regions of Tm that are critical for regulation. A broad internal region of Tm is important for its binding to actin decorated with myosin subfragment one (S1), and the strength of this binding correlates with the retention of physiological Tm-Tn-mediated regulation. These investigators have also studied the function of regions of 2-3 of Tm by deleting these regions
Biochemical and Biophysical Research Communications, 2000
We examined the effect of troponin I (TnI) phosphorylation by cAMP-dependent protein kinase (PKA) on the length-dependent tension activation in skinned rat cardiac trabeculae. Increasing sarcomere length shifted the pCa (؊log[Ca 2؉ ])-tension relation to the left. Treatment with PKA decreased the Ca 2؉ sensitivity of the myofilament and also decreased the lengthdependent shift of the pCa-tension relation. Replacement of endogenous TnI with phosphorylated TnI directly demonstrated that TnI phosphorylation is responsible for the decreased length-dependence. When MgATP concentration was lowered in the absence of Ca 2؉ , tension was elicited through rigorous crossbridge-induced thin filament activation. Increasing sarcomere length shifted the pMgATP (؊log[MgATP])tension relation to the right, and either TnI phosphorylation or partial extraction of troponin C (TnC) abolished this length-dependent shift. We conclude that TnI phosphorylation by PKA attenuates the lengthdependence of tension activation in cardiac muscle by decreasing the cross-bridge-dependent thin filament activation through a reduction of the interaction between TnI and TnC.
AJP: Heart and Circulatory Physiology, 2013
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, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50 (−log10 [Ca2+]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. pCa50 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 pCa50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+ sensitivity was blunted i...
Journal of Molecular and Cellular Cardiology, 2011
Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca(2+) binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca(2+)-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca(2+) dissociation rates (k(off)), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn&amp;gt;WT cTn&amp;gt;L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca(2+), L48Q cTnC did not affect maximum tension (T(max)), thin filament activation (k(ACT)) and tension development (k(TR)) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T(max), k(ACT) and k(TR) by 40-65% with little change in relaxation. Interestingly, k(ACT) was less than k(TR) with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca(2+)-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T(max), Ca(2+) sensitivity of tension (pCa(50)), and slope (n(H)) of tension-pCa, while L48Q cTn increased pCa(50) and reduced n(H). Increased cross-bridge cycling with 2-deoxy-ATP increased pCa(50) with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca(2+)-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2007
Coordinated expression of species-specific myosin heavy chain (MHC) and troponin (Tn) isoforms may bring about a dynamic complementarity to match muscle contraction speed with species-specific heart rates. Contractile system function and dynamic force-length measurements were made in muscle fibers from mouse and rat hearts and in muscle fibers after reconstitution with either recombinant homologous Tn or orthologous Tn. The rate constants of length-mediated cross-bridge (XB) recruitment ( b) and tension redevelopment ( ktr) of mouse fibers were significantly faster than those of rat fibers. Both the tension cost (ATPase/tension) and rate constant of length-mediated XB distortion ( c) were higher in the mouse than in the rat. Thus the mouse fiber was faster in all dynamic and functional aspects than the rat fiber. Mouse Tn significantly increased b and ktrin rat fibers; conversely, rat Tn significantly decreased b and ktrin mouse fibers. Thus the length-mediated recruitment of force-...