The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
Pieter P De Tombe - One of the best experts on this subject based on the ideXlab platform.
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cardiac resynchronization sensitizes the sarcomere to calcium by reactivating gsk 3β
Journal of Clinical Investigation, 2014Co-Authors: Jonathan A Kirk, Pieter P De Tombe, Wei Dong Gao, Viola Kooij, Ronald J Holewinski, Giulio Agnetti, Richard S Tunin, Namthip Witayavanitkul, Jennifer E Van Eyk, David A KassAbstract:Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance Myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances Myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of Myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β–dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of Myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.
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new insights into the functional significance of the acidic region of the unique n terminal extension of cardiac troponin i
Biochimica et Biophysica Acta, 2013Co-Authors: Marcus Henze, Pieter P De Tombe, Tomoyoshi Kobayashi, Stacey E Patrick, Aaron C Hinken, Sarah B Scruggs, Paul H Goldspink, Minae Kobayashi, Peipei Ping, John R. SolaroAbstract:Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-e (PKCe) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, Myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on Myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of Myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
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Interventricular differences in Myofilament function in experimental congestive heart failure
Pflügers Archiv - European Journal of Physiology, 2011Co-Authors: Rashad J. Belin, R. John Solaro, Marius P. Sumandea, Gail A. Sievert, Laura A. Harvey, David L. Geenen, Pieter P De TombeAbstract:This study was conducted to identify molecular mechanisms which explain interventricular differences in Myofilament function in experimental congestive heart failure (CHF). CHF was induced in rats by chronic aortic banding or myocardial infarction for 32–36 weeks. Right and left ventricular (RV, LV) myocytes were mechanically isolated, triton-skinned, and attached to a force transducer and motor arm. Myofilament force–[Ca^2+] relations assessed maximal Ca^2+-saturated force ( F _max) and the [Ca^2+] at 50% of F _max (EC_50). Myofilament protein phosphorylation was determined via ProQ diamond phospho-staining. Protein kinase C (PKC)-α expression/activation and site-specific phosphorylation of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were measured via immunoblotting. Relative to controls, failing RV myocytes displayed a ~45% decrease in F _max with no change in EC_50, whereas failing LV myocytes displayed a ~45% decrease in F _max and ~50% increase in EC_50. Failing LV Myofilaments were less Ca^2+-sensitive (37% increase in EC_50) than failing RV Myofilaments. Expression and activation of PKC-α was increased twofold in failing RV myocardium and relative to the RV, PKC-α was twofold higher in the failing LV, while PKC-β expression was unchanged by CHF. PKC-α-dependent phosphorylation and PP1-mediated dephosphorylation of failing RV Myofilaments increased EC_50 and increased F _max, respectively. Phosphorylation of cTnI and cTnT was greater in failing LV Myofilaments than in failing RV Myofilaments. RV Myofilament function is depressed in experimental CHF in association with increased PKC-α signaling and Myofilament protein phosphorylation. Furthermore, Myofilament dysfunction is greater in the LV compared to the RV due in part to increased PKC-α activation and phosphorylation of cTnI and cTnT.
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Myofilament length dependent activation
Journal of Molecular and Cellular Cardiology, 2010Co-Authors: Pieter P De Tombe, Gerrie P Farman, Ryan D. Mateja, Kittipong Tachampa, Younss Ait Mou, Thomas C IrvingAbstract:Abstract The Frank–Starling law of the heart describes the interrelationship between end-diastolic volume and cardiac ejection volume, a regulatory system that operates on a beat-to-beat basis. The main cellular mechanism that underlies this phenomenon is an increase in the responsiveness of cardiac Myofilaments to activating Ca2+ ions at a longer sarcomere length, commonly referred to as Myofilament length-dependent activation. This review focuses on what molecular mechanisms may underlie Myofilament length dependency. Specifically, the roles of inter-filament spacing, thick and thin filament based regulation, as well as sarcomeric regulatory proteins are discussed. Although the “Frank–Starling law of the heart” constitutes a fundamental cardiac property that has been appreciated for well over a century, it is still not known in muscle how the contractile apparatus transduces the information concerning sarcomere length to modulate ventricular pressure development.
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Cardiac thin filament regulation
Pflugers Archiv : European journal of physiology, 2008Co-Authors: Tomoyoshi Kobayashi, Lei Jin, Pieter P De TombeAbstract:Myocardial contraction is initiated upon the release of calcium into the cytosol from the sarcoplasmic reticulum following membrane depolarization. The fundamental physiological role of the heart is to pump an amount blood that is determined by the prevailing requirements of the body. The physiological control systems employed to accomplish this task include regulation of heart rate, the amount of calcium release, and the response of the cardiac Myofilaments to activator calcium ions. Thin filament activation and relaxation dynamics has emerged as a pivotal regulatory system tuning Myofilament function to the beat-to-beat regulation of cardiac output. Maladaptation of thin filament dynamics, in addition to dysfunctional calcium cycling, is now recognized as an important cellular mechanism causing reduced cardiac pump function in a variety of cardiac diseases. Here, we review current knowledge regarding protein-protein interactions involved in the dynamics of thin filament activation and relaxation and the regulation of these processes by protein kinase-mediated phosphorylation.
Eduardo Marbán - One of the best experts on this subject based on the ideXlab platform.
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transgenic mouse model of stunned myocardium
Science, 2000Co-Authors: Anne M Murphy, David A Kass, Jennifer E Van Eyk, Harald Kogler, Dimitrios Georgakopoulos, Jason L Mcdonough, Eduardo MarbánAbstract:Stunned myocardium is a syndrome of reversible contractile failure that frequently complicates coronary artery disease. Cardiac excitation is uncoupled from contraction at the level of the Myofilaments. Selective proteolysis of the thin filament protein troponin I has been correlated with stunned myocardium. Here, transgenic mice expressing the major degradation product of troponin I (TnI1-193) in the heart were found to develop ventricular dilatation, diminished contractility, and reduced Myofilament calcium responsiveness, recapitulating the phenotype of stunned myocardium. Proteolysis of troponin I also occurs in ischemic human cardiac muscle. Thus, troponin I proteolysis underlies the pathogenesis of a common acquired form of heart failure.
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novel Myofilament ca2 sensitizing property of xanthine oxidase inhibitors
Circulation Research, 1998Co-Authors: Nestor G Perez, Wei Dong Gao, Eduardo MarbánAbstract:Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia ("myocardial stunning"). Stunning decreases contractility at the level of the contractile proteins; therefore, we asked whether antioxidant treatment preserves Myofilament Ca2+ responsiveness after global ischemia and reflow. Right ventricular trabeculae were dissected from rat hearts subjected either to 20 minutes ischemia and reperfusion in the absence of drugs (stunned group) or to the same protocol in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), and mercaptopropionylglycine (MPG), a hydroxyl radical scavenger (antioxidant group). At 20 minutes of reflow, isovolumic developed pressure recovered completely in the antioxidant group, but in the stunned group it recovered by only 57%. [Ca2+]i and contractile force measurements in trabeculae revealed the expected depression of Myofilament function in the stunned group, with no change in Ca2+ transients relative to nonischemic controls. In contrast, Ca2+ transients were smaller, but force was greater, in the antioxidant group relative to both the stunned group and to nonischemic controls. Steady-state [Ca2+]i-force relationships revealed a striking increase of maximal force and a modest shift of activation to a lower range of [Ca2+]i. The increase in maximal force was reproduced by allopurinol+MPG or by allopurinol alone under nonischemic conditions and also by oxypurinol (100 micromol/L), a potent inhibitor of XO. We conclude that allopurinol and oxypurinol sensitize the cardiac Myofilaments to Ca2+. This Ca2+-sensitizing effect underlies the preservation of contractility observed with an allopurinol+MPG antioxidant cocktail in a model of stunned myocardium. These serendipitous findings identify allopurinol and oxypurinol as the lead compounds of a novel class of inotropic agents.
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Alterations of excitation—contraction coupling in stunned myocardium and in failing myocardium
Journal of Molecular and Cellular Cardiology, 1995Co-Authors: Dan Atar, Eduardo MarbánAbstract:D. Atar, W. D. Gao and E. Marban. Alterations of Excitation—Contraction Coupling in Stunned Myocardium and in Failing Myocardium. Journal of Molecular and Cellular Cardiology (1995) 27, 783–791. Although both myocardial stunning and chronic heart failure are characterized by contractile dysfunction, there are profound differences in their underlying mechanisms. Changes in cardiac contractile force can be effected by modulation of intracellular [Ca2+] or by alteration of the contractile protein response to intracellular Ca2+. New evidence suggests that the principal lesion in the stunned myocardium resides at the level of the contractile proteins, which may be injured by proteases activated early during reperfusion. In contrast, failing myocardium is known to display abnormal intracellular Ca2+ handling, indicative of dysfunction of the sarcoplasmic reticulum. Alterations of gene expression and isoform switching of the Myofilaments also occur in failing myocardium, consistent with an observed shift of the kinetics of crossbridge cycling. In conclusion, changes in both intracellular Ca2+ homeostasis and Myofilament function occur in failing myocardium, while stunned myocardium primarily reflects an uncoupling between Ca2+ and contractile force.
John R. Solaro - One of the best experts on this subject based on the ideXlab platform.
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new insights into the functional significance of the acidic region of the unique n terminal extension of cardiac troponin i
Biochimica et Biophysica Acta, 2013Co-Authors: Marcus Henze, Pieter P De Tombe, Tomoyoshi Kobayashi, Stacey E Patrick, Aaron C Hinken, Sarah B Scruggs, Paul H Goldspink, Minae Kobayashi, Peipei Ping, John R. SolaroAbstract:Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-e (PKCe) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, Myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on Myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of Myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
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Myofilament ca2 sensitization causes susceptibility to cardiac arrhythmia in mice
Journal of Clinical Investigation, 2008Co-Authors: Franz J. Baudenbacher, John R. Solaro, Tilmann Schober, Jose R Pinto, Veniamin Y Sidorov, Fredrick A Hilliard, James D Potter, Bjorn C. KnollmannAbstract:In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased Myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by Myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, Myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in Myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.
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augmented protein kinase c α induced Myofilament protein phosphorylation contributes to Myofilament dysfunction in experimental congestive heart failure
Circulation Research, 2007Co-Authors: Rashad J. Belin, Marius P. Sumandea, John R. Solaro, Edward Allen, Kelly Q Schoenfelt, Helen Wang, Pieter P De TombeAbstract:It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular Myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-alpha may be of particular importance. Here, we examined PKC-alpha quantity, activity, and signaling to Myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-alpha expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force-[Ca(2+)] relation to obtain Ca(2+)-saturated maximal force (F(max)) and Myofilament Ca(2+) sensitivity (indexed by EC(50)) before and after incubation with PKC-alpha, protein phosphatase type 1 (PP1), or PP2a. PKC-alpha treatment induced a 30% decline in F(max) and 55% increase in the EC(50) in control cells but had no impact on Myofilament function in failing cells. PP1-mediated dephosphorylation increased F(max) (15%) and decreased EC(50) ( approximately 20%) in failing Myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on Myofilament function in either group. Lastly, PP1 dephosphorylation restored Myofilament function in control cells hyperphosphorylated with PKC-alpha. Collectively, our results suggest that in end-stage congestive heart failure, the Myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-alpha.
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dilated cardiomyopathy mutant tropomyosin mice develop cardiac dysfunction with significantly decreased fractional shortening and Myofilament calcium sensitivity
Circulation Research, 2007Co-Authors: Sudarsan Rajan, John R. Solaro, Rafeeq P H Ahmed, Ganapathy Jagatheesan, Natalia Petrashevskaya, Greg P Boivin, Dalia Urboniene, Grace M Arteaga, Beata M Wolska, Stephen B LiggettAbstract:Mutations in striated muscle alpha-tropomyosin (alpha-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within alpha-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of alpha-TM mutations associated with DCM, we generated transgenic mice that express mutant alpha-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous alpha-TM levels, with total Myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the Myofilaments had significantly decreased Ca(2+) sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of beta-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca(2+) handling proteins sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor. Furthermore, our study also indicates that the alpha-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and Myofilament Ca(2+) sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.
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functional consequences of caspase activation in cardiac myocytes
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Catherine Communal, Pieter P De Tombe, Marius P. Sumandea, John R. Solaro, Jagat Narula, Roger J HajjarAbstract:Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac Myofilament including α-actin, α-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of α-actin and α-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac Myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using β-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca2+-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca2+ relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of Myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.
Olivier Cazorla - One of the best experts on this subject based on the ideXlab platform.
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Subendocardial Increase in Reactive Oxygen Species Production Affects Regional Contractile Function in Ischemic Heart Failure
Antioxidants and Redox Signaling, 2013Co-Authors: Lucas Andre, Alain Lacampagne, J. Fauconnier, Cyril Reboul, Christine Feillet Coudray, Pierre Meschin, Charlotte Farah, Gilles Fouret, Sylvain Richard, Olivier CazorlaAbstract:Aims: Heart failure (HF) is characterized by regionalized contractile alterations resulting in loss of the transmural contractile gradient across the left ventricular free wall. We tested whether a regional alteration in mitochondrial oxidative metabolism during HF could affect Myofilament function through protein kinase A (PKA) signaling. Results: Twelve weeks after permanent left coronary artery ligation that induced myocardial infarction (MI), subendocardial (Endo) cardiomyocytes had decreased activity of complex I and IV of the mitochondrial electron transport chain and produced twice more superoxide anions than sham Endo and subepicardial cells. This effect was associated with a reduced antioxidant activity of superoxide dismutase and Catalase only in MI Endo cells. The Myofilament contractile properties (Ca2+ sensitivity and maximal tension), evaluated in skinned cardiomyocytes, were also reduced only in MI Endo myocytes. Conversely, in MI rats treated with the antioxidant N-acetylcysteine (NAC) for 4 weeks, the generation of superoxide anions in Endo cardiomyocytes was normalized and the contractile properties of skinned cardiomyocytes restored. This effect was accompanied by improved in vivo contractility. The beneficial effects of NAC were mediated, at least, in part, through reduction of the PKA activity, which was higher in MI Myofilaments, particularly, the PKA-mediated hyperphosphorylation of cardiac Troponin I. Innovation: The Transmural gradient in the mitochondrial content/activity is lost during HF and mediates reactive oxygen species-dependent contractile dysfunction. Conclusions: Regionalized alterations in redox signaling affect the contractile machinery of sub-Endo myocytes through a PKA-dependent pathway that contributes to the loss of the transmural contractile gradient and impairs global contractility.
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Protein kinase D increases maximal Ca 2+ -activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser 315
AJP - Heart and Circulatory Physiology, 2012Co-Authors: Ellen Dirkx, Sakthivel Sadayappan, Olivier Cazorla, Johan Van Lint, Ilka Lorenzen-schmidt, Lucie Carrier, Guillaume J.j.m. Van Eys, Robert Schwenk, Jan Glatz, Joost J. F. P. LuikenAbstract:Dirkx E, Cazorla O, Schwenk RW, Lorenzen-Schmidt I, Sa-dayappan S, Van Lint J, Carrier L, van Eys GJ, Glatz JF, Luiken JJ. Protein kinase D increases maximal Ca 2ϩ-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser 315 .Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac Myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phos-phorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser 315. The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac Myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both Myofilament Ca 2ϩ sensitivity (pCa50) and maximal Ca 2ϩ-activated tension of contraction (Tmax). In cMyBP-C KO skinned myocytes, PKD increased pCa50 but did not alter Tmax. This suggests that cMyBP-C is not involved in PKD-mediated sensitization of Myofilaments to Ca 2ϩ but is essential for PKD-induced increase in Tmax. Furthermore, the phosphorylation of both PKD-Ser 916 and cMyBP-C-Ser 315 was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca 2ϩ sensitivity and by an increased Tmax through phosphorylation of cMyBP-C. cardiomyocyte contractility; calcium sensitivity; protein kinase A; phospho-cardiac myosin-binding protein C-serine-315
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Beneficial effects of SR33805 in failing myocardium
Cardiovascular Research, 2011Co-Authors: Younss Ait Mou, Alain Lacampagne, Attila Toth, Cécile Cassan, Daniel Czuriga, Pieter De Tombe, Zoltan Papp, Olivier CazorlaAbstract:Aims SR33805, a potent Ca 2+ channel blocker, increases cardiac Myofilament Ca 2+ sensitivity in healthy rat cardiomyo-cytes. Therefore, the aim of the present study was to evaluate the effects of SR33805 on contractile properties in ischaemic failing hearts after myocardial infarction (MI) in vivo and in vitro at the cellular level. Methods and results The effect of SR33805 (10 mM) was tested on the excitation-contraction coupling of cardiomyocytes isolated from rat with end-stage heart failure. Cell shortening and Ca 2+ transients were measured in intact cardiomyocytes, while contractile properties were determined in Triton X-100 permeabilized myocytes. Acute treatment with SR33805 restored the MI-altered cell shortening without affecting the Ca 2+ transient amplitude, suggesting an increase of myo-filament Ca 2+ sensitivity in MI myocytes. Indeed, a SR33805-induced sensitization of Myofilament activation was found to be associated with a slight increase in myosin light chain-2 phosphorylation and a more significant decrease on troponin I (TnI) phosphorylation. Decreased TnI phosphorylation was related to inhibition of protein kinase A activity by SR33805. Finally, administration of a single intra-peritoneal bolus of SR33805 (20 mg/kg) improved end-systolic strain and fractional shortening of MI hearts. Conclusion The present study indicates that treatment with SR33805 improved contractility of ischaemic failing hearts after MI in the rat by selectively modulating the phosphorylation status of sarcomeric regulatory proteins, which then sensitized the Myofilaments to Ca 2+. Our results gave a proof of concept that manipulation of the Ca 2+ sensitivity of sarco-meric regulatory proteins can be used to improve contractility of a failing heart.
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Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice
Cardiovascular Research, 2006Co-Authors: Olivier Cazorla, Alain Lacampagne, Szabolcs Szilagyi, Guy Vassort, Nicolas Vignier, Guillermo Salazar, Elisabeth Krämer, Lucie CarrierAbstract:Objective: h-Adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates proteins such as troponin I (cTnI) and C-protein (cMyBP-C). The relative contributions of cTnI and cMyBP-C to the regulation of Myofilament Ca 2+ sensitivity are still controversial because of difficulty in targeting specific protein phosphorylation. Recently, impaired relaxation was found in cMyBP-C-deficient mice (KO) in vivo under basal conditions and after h-adrenergic stimulation. The goal of this study was to analyse the length-dependent and PKA-dependent modulations of the cardiac contractile machinery in a mouse model lacking cMyBP-C. Methods: In the present work, we studied the PKA effect on Myofilament Ca 2+ sensitivity of left ventricular skinned myocytes isolated from 5-week-and 55-week-old wild-type (WT) and cMyBP-C knockout (KO) mice at 1.9 and 2.3 Am sarcomere lengths (SL). The cTnI content and phosphorylation status were examined by Western blot analysis. Results: Without PKA stimulation and at the shorter SL, Ca 2+ sensitivity was higher in KO compared to WT. The difference disappeared at the longer SL. No difference in passive tension or maximal active tension was observed. PKA stimulation induced a desensitization of WT Myofilaments at both SL but had almost no effect in KO Myofilaments despite similar levels of cTnI phosphorylation. We also observed expression of slow skeletal TnI in KO animals that was not correlated with the PKA effects. Conclusion: The results suggest that cMyBP-C contributes to the regulation of cardiac contraction at short sarcomere length and that Myofilament desensitization induced by PKA requires the presence of cMyBP-C and does not depend only upon TnI phosphorylation.
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Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte
Cardiovascular Research, 2006Co-Authors: Olivier Cazorla, Alain Lacampagne, Guy Vassort, Y Aït Mou, L Goret, M. Dauzat, S. Tanguy, P. ObertAbstract:Objective: Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. Methods: Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca 2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). Results: Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch-dependent Ca 2+ sensitization of the Myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca 2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca 2+ sensitivity. In contrast, high-altitude training decreased Ca 2+ sensitivity of the Myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca 2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. Conclusions: Training at sea level increased the transmural gradient of stretch-dependent Ca 2+ sensitization of the Myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed Myofilament response to Ca 2+ , especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism.
Thomas C Irving - One of the best experts on this subject based on the ideXlab platform.
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nanometer scale structure differences in the Myofilament lattice spacing of two cockroach leg muscles correspond to their different functions
The Journal of Experimental Biology, 2020Co-Authors: Travis Tune, Thomas C Irving, Simon SponbergAbstract:ABSTRACT Muscle is highly organized across multiple length scales. Consequently, small changes in the arrangement of Myofilaments can influence macroscopic mechanical function. Two leg muscles of a cockroach have identical innervation, mass, twitch responses, length–tension curves and force–velocity relationships. However, during running, one muscle is dissipative (a ‘brake’), while the other dissipates and produces significant positive mechanical work (bifunctional). Using time-resolved X-ray diffraction in intact, contracting muscle, we simultaneously measured the Myofilament lattice spacing, packing structure and macroscopic force production of these muscles to test whether structural differences in the Myofilament lattice might correspond to the muscles9 different mechanical functions. While the packing patterns are the same, one muscle has 1 nm smaller lattice spacing at rest. Under isometric stimulation, the difference in lattice spacing disappeared, consistent with the two muscles9 identical steady-state behavior. During periodic contractions, one muscle undergoes a 1 nm greater change in lattice spacing, which correlates with force. This is the first identified structural feature in the Myofilament lattice of these two muscles that shares their whole-muscle dynamic differences and quasi-static similarities.
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myocardial infarction induced n terminal fragment of cardiac myosin binding protein c cmybp c impairs Myofilament function in human myocardium
Journal of Biological Chemistry, 2014Co-Authors: Namthip Witayavanitkul, Younss Ait Mou, Jason Sarkey, Diederik W D Kuster, Ramzi J Khairallah, Suresh Govindan, Sudarsan Rajan, David F Wieczorek, Xinxin Chen, Thomas C IrvingAbstract:Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific Myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac Myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and Myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed Myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.
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myocardial infarction induced n terminal fragment of cmybp c impairs Myofilament function in human left ventricular myofibrils
Biophysical Journal, 2014Co-Authors: Namthip Witayavanitkul, Jason Sarkey, Younss Aitmou, Diederik W D Kuster, Ramzi J Khairallah, Suresh Govindan, Xin Chen, Sudarsan Rajan, David F Wieczorek, Thomas C IrvingAbstract:Rationale: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific Myofilament protein, is proteolyzed post-MI in humans and results in an N-terminal fragment, C0C1f. The presence of C0C1f in cultured adult cardiomyocytes results in decreased Ca2+ transients and cell shortening, in addition to the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear.Objective: To determine how C0C1f causes altered contractility in human cardiac Myofilaments in vitro.Methods and Results: We generated recombinant human C0C1f (hC0C1f) and incorporated it into skinned human left ventricular myocytes. Mechanical properties were then studied at sarcomere lengths of 2.0 and 2.3 µm. Our data demonstrate that the presence of hC0C1f in the sarcomere decreased maximal force Myofilament Ca2+ sensitivity, increased cooperative activation at short lengths and enhanced length-dependent activation. Furthermore, hC0C1f led to increased cross-bridge cycling kinetics and tension cost at both short and long sarcomere lengths. We further established that the detrimental effects of hC0C1f occur through direct interaction with the thin filament proteins actin and α-tropomyosin (α-TM).Conclusions: Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed Myofilament function and Ca2+ sensitivity in otherwise healthy human donor Myofilament preparations. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-TM, suggesting that cleaved C0C1f could act as a poison peptide and disrupt the interaction of native cMyBP-C with the thin filament.Keywords: Cross-bridge cycling kinetics; length-dependent activation; cMyBP-C; C0C1f protein.
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Myofilament length dependent activation
Journal of Molecular and Cellular Cardiology, 2010Co-Authors: Pieter P De Tombe, Gerrie P Farman, Ryan D. Mateja, Kittipong Tachampa, Younss Ait Mou, Thomas C IrvingAbstract:Abstract The Frank–Starling law of the heart describes the interrelationship between end-diastolic volume and cardiac ejection volume, a regulatory system that operates on a beat-to-beat basis. The main cellular mechanism that underlies this phenomenon is an increase in the responsiveness of cardiac Myofilaments to activating Ca2+ ions at a longer sarcomere length, commonly referred to as Myofilament length-dependent activation. This review focuses on what molecular mechanisms may underlie Myofilament length dependency. Specifically, the roles of inter-filament spacing, thick and thin filament based regulation, as well as sarcomeric regulatory proteins are discussed. Although the “Frank–Starling law of the heart” constitutes a fundamental cardiac property that has been appreciated for well over a century, it is still not known in muscle how the contractile apparatus transduces the information concerning sarcomere length to modulate ventricular pressure development.
