Vasco Sequeira

Perturbed Length-Dependent Activation in Human Hypertrophic Cardiomyopathy With Missense Sarcomeric Gene Mutations

Rationale: High-myofilament Ca2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca2+ sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca2+ sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. Conclusions: High-myofilament Ca2+ sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

The physiological role of cardiac cytoskeleton and its alterations in heart failure.

Cardiac muscle cells are equipped with specialized biochemical machineries for the rapid generation of force and movement central to the work generated by the heart. During each heart beat cardiac muscle cells perceive and experience changes in length and load, which reflect one of the fundamental principles of physiology known as the Frank-Starling law of the heart. Cardiac muscle cells are unique mechanical stretch sensors that allow the heart to increase cardiac output, and adjust it to new physiological and pathological situations. In the present review we discuss the mechano-sensory role of the cytoskeletal proteins with respect to their tight interaction with the sarcolemma and extracellular matrix. The role of contractile thick and thin filament proteins, the elastic protein titin, and their anchorage at the Z-disc and M-band, with associated proteins are reviewed in physiologic and pathologic conditions leading to heart failure. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

β‐adrenergic receptor signalling and its functional consequences in the diseased heart

To maintain the balance between the demand of the body and supply (cardiac output), cardiac performance is tightly regulated via the parasympathetic and sympathetic nervous systems. In heart failure, cardiac output (supply) is decreased due to pathologic remodelling of the heart. To meet the demands of the body, the sympathetic system is activated and catecholamines stimulate β‐adrenergic receptors (β‐ARs) to increase contractile performance and cardiac output. Although this is beneficial in the acute phase, chronic β‐ARs stimulation initiates a cascade of alterations at the cellular level, resulting in a diminished contractile performance of the heart.

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

Frank-Starlings 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.

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3&bgr; (GSK3&bgr;) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3&bgr; showed phosphorylation on Ser133. In addition, GSK3&bgr; phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3&bgr; treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3&bgr; phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3&bgr; at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

GSK3β Phosphorylates Newly Identified Site in the Pro-Ala Rich Region of Cardiac Myosin Binding Protein C and Alters Cross-Bridge Cycling Kinetics in Human

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3&bgr; (GSK3&bgr;) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3&bgr; showed phosphorylation on Ser133. In addition, GSK3&bgr; phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3&bgr; treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3&bgr; phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3&bgr; at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

Aims Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved. Methods and Results Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143. Conclusion PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.

Effect of levosimendan on the contractility of muscle fibers from nemaline myopathy patients with mutations in the nebulin gene

BackgroundNemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM.MethodsTo test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured.ResultsNo effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested.ConclusionsThese findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

Cardiac myosin-binding protein C: hypertrophic cardiomyopathy mutations and structure–function relationships

Cardiac myosin-binding protein C (cMyBP-C) research has been characterized by two waves. Initial interest was piqued by its discovery in 1973 as a contaminant of myosin preparations from skeletal muscle. The second wave started in 1995 by the discovery that mutations in the gene encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM). In this review, we will address what is known of cMyBP-Cs role as a regulator of contraction as well as its role in HCM.