Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range.

Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.

MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips.

Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca2+] conditions (pCa 8.0, where pCa = -log10[Ca2+]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I1,1/I1,0, also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca2+-activated force-pCa measurements show that Ca2+-sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca2+] and Ca2+-sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.

Increased myofilament calcium sensitivity is associated with decreased cardiac troponin I phosphorylation in the diabetic rat heart.

NEW FINDINGS: What is the central question of this study? In Zucker Diabetic Fatty rats, does cardiomyocyte myofilament function change through the time course of diabetes and what are the mechanisms behind alterations in calcium sensitivity? What is the main finding and its importance? Zucker Diabetic Fatty rats had increased myofilament calcium sensitivity and reduced phosphorylation at cardiac troponin I without differential O-GlcNAcylation. ABSTRACT: The diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament calcium regulation of contraction is altered while also categorizing the levels of phosphorylation and O-GlcNAcylation in the myofilaments. Calcium sensitivity (pCa50 ) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12 weeks old) and after 8 weeks of uncontrolled hyperglycaemia (20 weeks old) and in non-diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+ ]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho-sites on cardiac troponin I (cTnI) and total cTnI O-GlcNAcylation were quantified using immunoblot. pCa50 was greater in both 12- and 20-week-old diabetic (DM) rats compared to nDM littermates (P = 0.0001). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (P = 0.003 and P = 0.01, respectively), but cTnI O-GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats and suggests an important role for cTnI phosphorylation in the DM heart.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment.

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Myofilament Ca2+ sensitivity correlates with left ventricular contractility during the progression of pressure overload-induced left ventricular myocardial hypertrophy in rats.

AIM: Here we aimed at investigating the relation between left ventricular (LV) contractility and myofilament function during the development and progression of pressure overload (PO)-induced LV myocardial hypertrophy (LVH). METHODS: Abdominal aortic banding (AB) was performed to induce PO in rats for 6, 12 and 18 weeks. Sham operated animals served as controls. Structural and molecular alterations were investigated by serial echocardiography, histology, quantitative real-time PCR and western blot. LV function was assessed by pressure-volume analysis. Force measurement was carried out in permeabilized cardiomyocytes. RESULTS: AB resulted in the development of pathological LVH as indicated by increased heart weight-to-tibial length ratio, LV mass index, cardiomyocyte diameter and fetal gene expression. These alterations were already present at early stage of LVH (AB-week6). Furthermore, at more advanced stages (AB-week12, AB-week18), myocardial fibrosis and chamber dilatation were also observed. From a hemodynamic point of view, the AB-wk6 group was associated with increased LV contractility, maintained ventriculo-arterial coupling (VAC) and preserved systolic function. In the same experimental group, increased myofilament Ca2+ sensitivity (pCa50) and hyperphosphorylation of cardiac troponin-I (cTnI) at Threonine-144 was detected. In contrast, in the AB-wk12 and AB-wk18 groups, the initial augmentation of LV contractility, as well as the increased myofilament Ca2+ sensitivity and cTnI (Threonine-144) hyperphosphorylation diminished, leading to impaired VAC and reduced systolic performance. Strong correlation was found between LV contractility parameters and myofilament Ca2+-sensitivity among the study groups. CONCLUSION: Changes in myofilament Ca2+ sensitivity might underlie the alterations in LV contractility during the development and progression of PO-induced LVH.

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis.

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 µm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 µm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.

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.

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca(2+)-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 Ca(2+)-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 Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

Downsizing the molecular spring of the giant protein titin reveals that skeletal muscle titin determines passive stiffness and drives longitudinal hypertrophy.

Titin, the largest protein known, forms an elastic myofilament in the striated muscle sarcomere. To establish titin's contribution to skeletal muscle passive stiffness, relative to that of the extracellular matrix, a mouse model was created in which titin's molecular spring region was shortened by deleting 47 exons, the TtnΔ112-158 model. RNA sequencing and super-resolution microscopy predicts a much stiffer titin molecule. Mechanical studies with this novel mouse model support that titin is the main determinant of skeletal muscle passive stiffness. Unexpectedly, the in vivo sarcomere length working range was shifted to shorter lengths in TtnΔ112-158 mice, due to a ~ 30% increase in the number of sarcomeres in series (longitudinal hypertrophy). The expected effect of this shift on active force generation was minimized through a shortening of thin filaments that was discovered in TtnΔ112-158 mice. Thus, skeletal muscle titin is the dominant determinant of physiological passive stiffness and drives longitudinal hypertrophy. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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 Ca(2+)-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (k rel), and recruitment (k df) due to rapid stretch, as well as the rate of force redevelopment (k tr) 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 k tr, measured at half-maximal Ca(2+)-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, k rel and k df were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, k tr 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 k rel and k df. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.