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Effects of Protein Kinase A on the Phosphorylation Status and Transverse Stiffness of Cardiac Myofibrils
J Pharmacol Sci 123, 279 – 283 (2013)
Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Short Communication
Effects of Protein Kinase A on the Phosphorylation Status and Transverse Stiffness of Cardiac Myofibrils Yoshiki Ohnuki1, Takenori Yamada2, Yasumasa Mototani1, Daisuke Umeki3, Kouichi Shiozawa1, Takayuki Fujita4, Yasutake Saeki1, and Satoshi Okumura1,4,* Department of Physiology, 3Department of Orthodontics, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan 2 Department of Physics (Biophysics Section), Faculty of Science, Tokyo University of Science, Tokyo 162-8601, Japan 4 Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan 1
Received July 3, 2013; Accepted September 2, 2013
Abstract. Stimulation of b-adrenergic receptors in cardiac myocytes activates cyclic AMP– dependent protein kinase A (PKA). PKA-mediated phosphorylation of myofibrils decreases their longitudinal stiffness, but its effect on transverse stiffness is not fully understood. We thus examined the effects of PKA treatment on the transverse stiffness of cardiac myofibrils by atomic force microscopy and determined the phosphorylation levels of myofibril components by SDSPAGE. Transverse stiffness was significantly decreased by PKA treatment concomitantly with increased phosphorylation of troponin I, myosin-binding protein C, and titin (also called connectin). Subsequent treatment with protein phosphatase 1 abrogated these PKA-mediated effects. [Supplementary methods: available only at http://dx.doi.org/10.1254/jphs.13110SC] Keywords: stiffness, catecholamine, contractility
play an important role in accelerating cross-bridge cycling and in reducing the Ca2+-sensitivity of the force, which may in turn contribute to the passive longitudinal stiffness (5). The giant myofibril component titin has been recognized as a major determinant of myocardial passive tension and PKA-mediated phosphorylation of titin results in a decrease in resting tension (6 – 8). However, the effect of b-AR stimulation on the transverse stiffness has not been well characterized. In this study, we examined the effects of PKA-mediated posttranslational modification on the phosphorylation levels of cardiac myofibril components by means of SDS-PAGE, and on the transverse stiffness of single myofibrils by means of AFM. Further details of the method can be found in the Supplementary methods (available in the online version only). The transverse stiffness of single cardiac myofibrils was measured in a relaxed state after PKA or PKA plus subsequent protein phosphatase 1 (PP1) treatment (PKA + PP1) by means of AFM (9) (Fig. 1B). Transverse stiffness was slightly but significantly decreased by PKA treatment for 30 min, but subsequent PP1 treatment abrogated the PKA-mediated decrease. It might be pos-
The atomic force microscope (AFM) can be applied to study the elasticity of various biological materials based on the deflection of the cantilever produced by movement of the tip across the specimen. Actomyosin cross-bridges have components of stiffness and force that are directed both longitudinally and transversely with respect to the filament axis (1). Although changes of longitudinal stiffness have been investigated, changes of transverse stiffness have not been well characterized. Here, we used AFM to examine changes in the transverse stiffness of cardiac myofibrils under physiological conditions (Fig. 1A). Stimulation of b-adrenergic receptors (b-AR) in cardiac myocytes activates cAMP-dependent protein kinase A (PKA) via activation of adenylyl cyclase (2 – 4), and PKA-mediated posttranslational modifications of myofibril components, such as troponin I (TnI), myosin binding protein-C (MyBP-C), and titin (also called connectin), have recently been demonstrated to *Corresponding author. [email protected] Published online in J-STAGE on October 22, 2013 doi: 10.1254/jphs.13110SC
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Fig. 1. Effects of PKA on the transverse stiffness of single cardiac myofibrils. A) The AFM cantilever is positioned in gentle contact with a myofibril, and slides along a single myofibril to measure the transverse stiffness (upper). Bright-field images of the AFM cantilever positioned over a myofibril (lower). B) Average transverse stiffness of single myofibrils before (Control) and after PKA or PKA plus subsequent PP1 treatment (PKA + PP1) (**P < 0.01 vs. Control, ††P < 0.01 vs. PKA, n = 8). C) Average transverse stiffness of single myofibrils before (Control) and after PP1 or PP1 plus subsequent PKA treatment (PP1 + PKA) [P = NS (not significant) vs. Control, ††P < 0.01 vs. PKA, n = 4].
sible to obtain a greater decrease of transverse stiffness by increasing the incubation time with PKA to ensure maximal phosphorylation of myofibrils (7). We also examined the transverse stiffness in the relaxed state after PP1 or PP1 plus subsequent PKA (Fig. 1C). Transverse stiffness tended to be increased by PP1 treatment, but the difference was not significant. However, subsequent PKA treatment significantly decreased the transverse stiffness of the PP1-treated myofibrils. These data indicate that PKA-mediated myofibril phosphorylation may modulate the decrease of transverse stiffness. We next examined the phosphorylation status of the components of the cardiac myofibrils (Fig. 2) (10). PKA treatment significantly increased the phosphorylation levels of TnI (2.0-fold), titin (2.1-fold), and MyBP-C (1.5-fold). We also examined the phosphorylation levels of each myofibril component (Fig. 3) in the relaxed state after PP1 or PP1 plus subsequent PKA treatment. PP1 alone tended to decrease the myofibril phosphoryla-
tion levels, but the changes were not significant. However, subsequent PKA treatment significantly increased the phosphorylation of TnI (1.8-fold), titin (1.7-fold), and MyBP-C (1.5-fold). Therefore, phosphorylation of titin, a major modulator of the passive longitudinal stiffness, as well as TnI and MyBP-C, might be involved in muscle relaxation by causing a decrease in the transverse stiffness of myocardial fibers in response to b-AR stimulation under normal physiological conditions (11, 12). Two PKA-mediated phosphorylation bands of titin: N2B-titin band, an intact rat cardiac isoform, and T2-titin band, a proteolytic fragment of the N2B-titin isoform, were observed (Figs. 2C and 3C) (8). It might be possible to obtain a greater decrease of transverse stiffness by decreasing the T2 degradation product under another preferable experimental condition. Heart failure is a common condition associated with high morbidity and mortality rates. Most research has focused on contractile dysfunction, but has essentially
Myofibrils and Transverse Stiffness
281
Fig. 2. Effects of PKA on the phosphorylation status of the cardiac myofibrils. A, C) Typical SDS-PAGE pattern of myofibril components: myosin light chain 2 (MLC2), troponin I (TnI), troponin T (TnT) myosin-binding protein C (MyBP-C), and titin (connectin). The N2B-titin band is an intact cardiac isoform and the T2-titin band is a proteolytic fragment of the N2B-titin isoform. The gel was stained with Pro-Q Diamond (specific for phosphorylated proteins) and subsequently stained with SYPRO Ruby (for total proteins). B, D) Relative phosphorylation levels of cardiac myofibril components. (*P < 0.05 or **P < 0.01 vs. Control, #P < 0.05 or ##P < 0.05 vs. PKA, P = NS vs. Control, n = 5).
ignored the importance of the post-translational modifications of myocardial fibers which may be responsible for the myocardial stiffness and diastolic dysfunction. Actomyosin cross-bridges have components of stiffness and force that are directed both longitudinally and transversely with respect to the filament axis (1). Although changes of longitudinal stiffness have been investigated, changes of transverse stiffness have not been well characterized. Our results suggest that dysfunction in the modulation of the transverse stiffness of myocardial fibers via PKA-mediated phosphorylation of fiber components could also be an important factor in heart failure. Previous findings on length-dependent activation following PKA treatment are inconclusive: it has been reported to be increased (13), unchanged (14), or decreased (15). The effect of decreased transverse
stiffness on the length-dependent activation is unknown, but in order to improve heart failure therapy it may be necessary to clarify the effect of the decreased transverse stiffness following PKA treatment on the lengthdependent activation by AFM. Acknowledgments This study was supported in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology (S.O., Y.M., T.F.); Takeda Science Foundation (S.O.); Yokohama Foundation for Advancement of Medical Science (S.O., T.F.); Mitsubishi Pharma Research Foundation (S.O.); Research for Promoting Technological Seeds A (discovery type) (S.O.); Yokohama Academic Foundation (Y.O., S.O.); 2010 Commercialization Promotion Program for Biotechnology-related Studies (S.O.); Grant for Research and Development Project II of Yokohama City University (S.O.).
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Fig. 3. Effects of PP1 on the phosphorylation status of the cardiac myofibrils. A, C) Typical SDS-PAGE pattern of cardiac myofibril components: myosin light chain 2 (MLC2), troponin I (TnI), troponin T (TnT) myosin-binding protein C (MyBP-C), and titin (connectin). The N2B-titin band is an intact cardiac isoform and the T2-titin band is a proteolytic fragment of the N2Btitin isoform. The gel was stained with Pro-Q Diamond (specific for phosphorylated proteins) and subsequently stained with SYPRO Ruby (for total proteins). B, D) Relative phosphorylation levels of cardiac myofibril components. (#P < 0.05 or ##P < 0.01 vs. PP1, P = NS vs. Control, n = 5).
References 1 Sumita Yoshikawa W, Nakamura K, Miura D, Shimizu J, Hashimoto K, Kataoka N, et al. Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic b-adrenergic stimulation. Circ J. 2012;77: 741–748. 2 Okumura S, Kawabe J, Yatani A, Takagi G, Lee MC, Hong C, et al. Type 5 adenylyl cyclase disruption alters not only sympathetic but also parasympathetic and calcium-mediated cardiac regulation. Circ Res. 2003;93:364–371. 3 Okumura S, Vatner DE, Kurotani R, Bai Y, Gao S, Yuan Z, et al. Disruption of type 5 adenylyl cyclase enhances desensitization of cyclic adenosine monophosphate signal and increases Akt signal with chronic catecholamine stress. Circulation. 2007;116: 1776–1783. 4 Okumura S, Suzuki S, Ishikawa Y. New aspects for the treatment of cardiac diseases based on the diversity of functional controls on cardiac muscles: effects of targeted disruption of the type 5
adenylyl cyclase gene. J Pharmacol Sci. 2009;109:354–359. 5 Gautel M, Zuffardi O, Freiburg A, Labeit S. Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J. 1995;14:1952–1960. 6 Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H. Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circ Res. 2002;90:1181–1188. 7 Fukuda N, Wu Y, Nair P, Granzier HL. Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoformdependent manner. J Gen Physiol. 2005;125:257–271. 8 Kruger M, Linke WA. Protein kinase-A phosphorylates titin in human heart muscle and reduces myofibrillar passive tension. J Muscle Res Cell Motil. 2006;27:435–444. 9 Akiyama N, Ohnuki Y, Kunioka Y, Saeki Y, Yamada T. Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy. J Physiol Sci. 2006;56: 145–151. 10 Ohnuki Y, Nishimura S, Sugiura S, Saeki Y. Phosphorylation
Myofibrils and Transverse Stiffness status of regulatory proteins and functional characteristics in myocardium of dilated cardiomyopathy of Syrian hamsters. J Physiol Sci. 2008;58:15–20. 11 van Heerebeek L, Franssen CP, Hamdani N, Verheugt FW, Somsen GA, Paulus WJ. Molecular and cellular basis for diastolic dysfunction. Curr Heart Fail Rep. 2012;9:293–302. 12 Palmer BM, Sadayappan S, Wang Y, Weith AE, Previs MJ, Bekyarova T, et al. Roles for cardiac MyBP-C in maintaining myofilament lattice rigidity and prolonging myosin cross-bridge lifetime. Biophys J. 2011;101:1661–1669. 13 Konhilas JP, Irving TC, Wolska BM, Jweied EE, Martin AF, Solaro RJ, et al. Troponin I in the murine myocardium: influence
283
on length-dependent activation and interfilament spacing. J Physiol. 2003;547:951–961. 14 van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res. 2003; 57:37–47. 15 Kajiwara H, Morimoto S, Fukuda N, Ohtsuki I, Kurihara S. Effect of troponin I phosphorylation by protein kinase A on length-dependence of tension activation in skinned cardiac muscle fibers. Biochem Biophys Res Commun. 2000;272:104–110.
Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Short Communication
Effects of Protein Kinase A on the Phosphorylation Status and Transverse Stiffness of Cardiac Myofibrils Yoshiki Ohnuki1, Takenori Yamada2, Yasumasa Mototani1, Daisuke Umeki3, Kouichi Shiozawa1, Takayuki Fujita4, Yasutake Saeki1, and Satoshi Okumura1,4,* Department of Physiology, 3Department of Orthodontics, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan 2 Department of Physics (Biophysics Section), Faculty of Science, Tokyo University of Science, Tokyo 162-8601, Japan 4 Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan 1
Received July 3, 2013; Accepted September 2, 2013
Abstract. Stimulation of b-adrenergic receptors in cardiac myocytes activates cyclic AMP– dependent protein kinase A (PKA). PKA-mediated phosphorylation of myofibrils decreases their longitudinal stiffness, but its effect on transverse stiffness is not fully understood. We thus examined the effects of PKA treatment on the transverse stiffness of cardiac myofibrils by atomic force microscopy and determined the phosphorylation levels of myofibril components by SDSPAGE. Transverse stiffness was significantly decreased by PKA treatment concomitantly with increased phosphorylation of troponin I, myosin-binding protein C, and titin (also called connectin). Subsequent treatment with protein phosphatase 1 abrogated these PKA-mediated effects. [Supplementary methods: available only at http://dx.doi.org/10.1254/jphs.13110SC] Keywords: stiffness, catecholamine, contractility
play an important role in accelerating cross-bridge cycling and in reducing the Ca2+-sensitivity of the force, which may in turn contribute to the passive longitudinal stiffness (5). The giant myofibril component titin has been recognized as a major determinant of myocardial passive tension and PKA-mediated phosphorylation of titin results in a decrease in resting tension (6 – 8). However, the effect of b-AR stimulation on the transverse stiffness has not been well characterized. In this study, we examined the effects of PKA-mediated posttranslational modification on the phosphorylation levels of cardiac myofibril components by means of SDS-PAGE, and on the transverse stiffness of single myofibrils by means of AFM. Further details of the method can be found in the Supplementary methods (available in the online version only). The transverse stiffness of single cardiac myofibrils was measured in a relaxed state after PKA or PKA plus subsequent protein phosphatase 1 (PP1) treatment (PKA + PP1) by means of AFM (9) (Fig. 1B). Transverse stiffness was slightly but significantly decreased by PKA treatment for 30 min, but subsequent PP1 treatment abrogated the PKA-mediated decrease. It might be pos-
The atomic force microscope (AFM) can be applied to study the elasticity of various biological materials based on the deflection of the cantilever produced by movement of the tip across the specimen. Actomyosin cross-bridges have components of stiffness and force that are directed both longitudinally and transversely with respect to the filament axis (1). Although changes of longitudinal stiffness have been investigated, changes of transverse stiffness have not been well characterized. Here, we used AFM to examine changes in the transverse stiffness of cardiac myofibrils under physiological conditions (Fig. 1A). Stimulation of b-adrenergic receptors (b-AR) in cardiac myocytes activates cAMP-dependent protein kinase A (PKA) via activation of adenylyl cyclase (2 – 4), and PKA-mediated posttranslational modifications of myofibril components, such as troponin I (TnI), myosin binding protein-C (MyBP-C), and titin (also called connectin), have recently been demonstrated to *Corresponding author. [email protected] Published online in J-STAGE on October 22, 2013 doi: 10.1254/jphs.13110SC
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Fig. 1. Effects of PKA on the transverse stiffness of single cardiac myofibrils. A) The AFM cantilever is positioned in gentle contact with a myofibril, and slides along a single myofibril to measure the transverse stiffness (upper). Bright-field images of the AFM cantilever positioned over a myofibril (lower). B) Average transverse stiffness of single myofibrils before (Control) and after PKA or PKA plus subsequent PP1 treatment (PKA + PP1) (**P < 0.01 vs. Control, ††P < 0.01 vs. PKA, n = 8). C) Average transverse stiffness of single myofibrils before (Control) and after PP1 or PP1 plus subsequent PKA treatment (PP1 + PKA) [P = NS (not significant) vs. Control, ††P < 0.01 vs. PKA, n = 4].
sible to obtain a greater decrease of transverse stiffness by increasing the incubation time with PKA to ensure maximal phosphorylation of myofibrils (7). We also examined the transverse stiffness in the relaxed state after PP1 or PP1 plus subsequent PKA (Fig. 1C). Transverse stiffness tended to be increased by PP1 treatment, but the difference was not significant. However, subsequent PKA treatment significantly decreased the transverse stiffness of the PP1-treated myofibrils. These data indicate that PKA-mediated myofibril phosphorylation may modulate the decrease of transverse stiffness. We next examined the phosphorylation status of the components of the cardiac myofibrils (Fig. 2) (10). PKA treatment significantly increased the phosphorylation levels of TnI (2.0-fold), titin (2.1-fold), and MyBP-C (1.5-fold). We also examined the phosphorylation levels of each myofibril component (Fig. 3) in the relaxed state after PP1 or PP1 plus subsequent PKA treatment. PP1 alone tended to decrease the myofibril phosphoryla-
tion levels, but the changes were not significant. However, subsequent PKA treatment significantly increased the phosphorylation of TnI (1.8-fold), titin (1.7-fold), and MyBP-C (1.5-fold). Therefore, phosphorylation of titin, a major modulator of the passive longitudinal stiffness, as well as TnI and MyBP-C, might be involved in muscle relaxation by causing a decrease in the transverse stiffness of myocardial fibers in response to b-AR stimulation under normal physiological conditions (11, 12). Two PKA-mediated phosphorylation bands of titin: N2B-titin band, an intact rat cardiac isoform, and T2-titin band, a proteolytic fragment of the N2B-titin isoform, were observed (Figs. 2C and 3C) (8). It might be possible to obtain a greater decrease of transverse stiffness by decreasing the T2 degradation product under another preferable experimental condition. Heart failure is a common condition associated with high morbidity and mortality rates. Most research has focused on contractile dysfunction, but has essentially
Myofibrils and Transverse Stiffness
281
Fig. 2. Effects of PKA on the phosphorylation status of the cardiac myofibrils. A, C) Typical SDS-PAGE pattern of myofibril components: myosin light chain 2 (MLC2), troponin I (TnI), troponin T (TnT) myosin-binding protein C (MyBP-C), and titin (connectin). The N2B-titin band is an intact cardiac isoform and the T2-titin band is a proteolytic fragment of the N2B-titin isoform. The gel was stained with Pro-Q Diamond (specific for phosphorylated proteins) and subsequently stained with SYPRO Ruby (for total proteins). B, D) Relative phosphorylation levels of cardiac myofibril components. (*P < 0.05 or **P < 0.01 vs. Control, #P < 0.05 or ##P < 0.05 vs. PKA, P = NS vs. Control, n = 5).
ignored the importance of the post-translational modifications of myocardial fibers which may be responsible for the myocardial stiffness and diastolic dysfunction. Actomyosin cross-bridges have components of stiffness and force that are directed both longitudinally and transversely with respect to the filament axis (1). Although changes of longitudinal stiffness have been investigated, changes of transverse stiffness have not been well characterized. Our results suggest that dysfunction in the modulation of the transverse stiffness of myocardial fibers via PKA-mediated phosphorylation of fiber components could also be an important factor in heart failure. Previous findings on length-dependent activation following PKA treatment are inconclusive: it has been reported to be increased (13), unchanged (14), or decreased (15). The effect of decreased transverse
stiffness on the length-dependent activation is unknown, but in order to improve heart failure therapy it may be necessary to clarify the effect of the decreased transverse stiffness following PKA treatment on the lengthdependent activation by AFM. Acknowledgments This study was supported in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology (S.O., Y.M., T.F.); Takeda Science Foundation (S.O.); Yokohama Foundation for Advancement of Medical Science (S.O., T.F.); Mitsubishi Pharma Research Foundation (S.O.); Research for Promoting Technological Seeds A (discovery type) (S.O.); Yokohama Academic Foundation (Y.O., S.O.); 2010 Commercialization Promotion Program for Biotechnology-related Studies (S.O.); Grant for Research and Development Project II of Yokohama City University (S.O.).
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Y Ohnuki et al
Fig. 3. Effects of PP1 on the phosphorylation status of the cardiac myofibrils. A, C) Typical SDS-PAGE pattern of cardiac myofibril components: myosin light chain 2 (MLC2), troponin I (TnI), troponin T (TnT) myosin-binding protein C (MyBP-C), and titin (connectin). The N2B-titin band is an intact cardiac isoform and the T2-titin band is a proteolytic fragment of the N2Btitin isoform. The gel was stained with Pro-Q Diamond (specific for phosphorylated proteins) and subsequently stained with SYPRO Ruby (for total proteins). B, D) Relative phosphorylation levels of cardiac myofibril components. (#P < 0.05 or ##P < 0.01 vs. PP1, P = NS vs. Control, n = 5).
References 1 Sumita Yoshikawa W, Nakamura K, Miura D, Shimizu J, Hashimoto K, Kataoka N, et al. Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic b-adrenergic stimulation. Circ J. 2012;77: 741–748. 2 Okumura S, Kawabe J, Yatani A, Takagi G, Lee MC, Hong C, et al. Type 5 adenylyl cyclase disruption alters not only sympathetic but also parasympathetic and calcium-mediated cardiac regulation. Circ Res. 2003;93:364–371. 3 Okumura S, Vatner DE, Kurotani R, Bai Y, Gao S, Yuan Z, et al. Disruption of type 5 adenylyl cyclase enhances desensitization of cyclic adenosine monophosphate signal and increases Akt signal with chronic catecholamine stress. Circulation. 2007;116: 1776–1783. 4 Okumura S, Suzuki S, Ishikawa Y. New aspects for the treatment of cardiac diseases based on the diversity of functional controls on cardiac muscles: effects of targeted disruption of the type 5
adenylyl cyclase gene. J Pharmacol Sci. 2009;109:354–359. 5 Gautel M, Zuffardi O, Freiburg A, Labeit S. Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J. 1995;14:1952–1960. 6 Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H. Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circ Res. 2002;90:1181–1188. 7 Fukuda N, Wu Y, Nair P, Granzier HL. Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoformdependent manner. J Gen Physiol. 2005;125:257–271. 8 Kruger M, Linke WA. Protein kinase-A phosphorylates titin in human heart muscle and reduces myofibrillar passive tension. J Muscle Res Cell Motil. 2006;27:435–444. 9 Akiyama N, Ohnuki Y, Kunioka Y, Saeki Y, Yamada T. Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy. J Physiol Sci. 2006;56: 145–151. 10 Ohnuki Y, Nishimura S, Sugiura S, Saeki Y. Phosphorylation
Myofibrils and Transverse Stiffness status of regulatory proteins and functional characteristics in myocardium of dilated cardiomyopathy of Syrian hamsters. J Physiol Sci. 2008;58:15–20. 11 van Heerebeek L, Franssen CP, Hamdani N, Verheugt FW, Somsen GA, Paulus WJ. Molecular and cellular basis for diastolic dysfunction. Curr Heart Fail Rep. 2012;9:293–302. 12 Palmer BM, Sadayappan S, Wang Y, Weith AE, Previs MJ, Bekyarova T, et al. Roles for cardiac MyBP-C in maintaining myofilament lattice rigidity and prolonging myosin cross-bridge lifetime. Biophys J. 2011;101:1661–1669. 13 Konhilas JP, Irving TC, Wolska BM, Jweied EE, Martin AF, Solaro RJ, et al. Troponin I in the murine myocardium: influence
283
on length-dependent activation and interfilament spacing. J Physiol. 2003;547:951–961. 14 van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res. 2003; 57:37–47. 15 Kajiwara H, Morimoto S, Fukuda N, Ohtsuki I, Kurihara S. Effect of troponin I phosphorylation by protein kinase A on length-dependence of tension activation in skinned cardiac muscle fibers. Biochem Biophys Res Commun. 2000;272:104–110.