Pieter Tombe - Academia.edu (original) (raw)

Papers by Pieter Tombe

Research paper thumbnail of Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

Pflügers Archiv - European Journal of Physiology, 2008

The present study examined the contribution of myofilament contractile proteins to regional funct... more The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca 2+ sensitivity, and cross-bridge cycling kinetics (K tr ) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca 2+ sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K tr was unchanged by stretch, but was higher in EPI cells possibly due to a higher α-MHC content. Submaximal Ca 2+ -activated K tr increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosinbinding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.

Research paper thumbnail of Beneficial effects of SR33805 in failing myocardium

Cardiovascular Research, 2011

1 This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca 2+... more 1 This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca 2+antagonistic properties, on the contractile properties of intact and skinned rat ventricular myocytes. 2 On intact cells loaded with the Ca 2+ -fluorescent indicator Indo-1, the application of low concentrations of SR33805 enhanced the amplitude of unloaded cell shortening and decreased the duration of cell shortening. Amplitude of the Ca 2+ transient was also decreased. 3 These effects were accompanied with a shortening of the action potential and a dose-dependent blockade of L-type calcium current (IC 50 ¼ 2.4 Â 10 À8 M). 4 On skinned cardiac cells, the application of a low SR33805 concentration (10 À8 M) induced a significant increase in maximal Ca 2+ -activated force at the two-tested sarcomere lengths (SLs), 1.9 and 2.3 mm. 5 The application of a larger dose of SR33805 (10 À6 -10 À5 M) induced a significant leftward shift of the tension -pCa relation that accounts for Ca 2+ -sensitization of the myofilaments, particularly at 2.3 mm SL. 6 In conclusion, despite its strong Ca 2+ -antagonistic properties SR33805 increases cardiac cell contractile activity as a consequence of its Ca 2+ -sensitizing effects. These effects are attributable to both an increase in the maximal Ca 2+ -activated force and a length-dependent Ca 2+ -sensitization.

Research paper thumbnail of Activation Cardiac Troponin I Threonine 144: Role in Myofilament LengthDependent

Myofilament length-dependent activation is the main cellular mechanism responsible for the Frank-... more Myofilament length-dependent activation is the main cellular mechanism responsible for the Frank-Starling law of the heart. All striated muscle display lengthdependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length-dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length-dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnI Thr>Pro and ssTnI Pro>Thr . Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force-Ca 2؉ relationships were determined to derive myofilament Ca 2؉ sensitivity (EC 50 ) at 2 sarcomere lengths: 2.0 and 2.2 m (n‫.)7؍‬ Myofilament lengthdependent activation was indexed as ⌬EC 50 , the difference in EC 50 between sarcomere lengths of 2.0 and 2.2 m. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced ⌬EC 50 from 1.26؎0.30 to 0.19؎0.04 mol/L. A similar reduction also could be observed when Tn contained cTnI Thr>Pro (⌬EC 50 ‫40.0؎42.0؍‬ mol/L), whereas the presence of ssTnI Pro>Thr increased ⌬EC 50 to 0.94؎0.12 mol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length-dependent activation.

Research paper thumbnail of cGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation: A Tale of Two Isoforms

Research paper thumbnail of Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing

The Journal of Physiology, 2003

Research paper thumbnail of Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

Pflügers Archiv - European Journal of Physiology, 2008

The present study examined the contribution of myofilament contractile proteins to regional funct... more The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca 2+ sensitivity, and cross-bridge cycling kinetics (K tr ) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca 2+ sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K tr was unchanged by stretch, but was higher in EPI cells possibly due to a higher α-MHC content. Submaximal Ca 2+ -activated K tr increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosinbinding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.

Research paper thumbnail of Blebbistatin: use as inhibitor of muscle contraction

Pflügers Archiv - European Journal of Physiology, 2008

Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In... more Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In this study, we tested BLEB in intact and skinned isolated rat cardiac trabeculae, rat intact myocytes, and single rabbit psoas myofibrils. BLEB (10 muM) reduced twitch force and cell shortening that was reversed by exposure to light. BLEB treatment of skinned trabeculae in the dark (1 hr) reduced Ca(2+)-activated force (EC(50) = 0.38 +/- 0.03 muM). Rapid (<5 ms) BLEB application in Ca(2+)-activated rabbit myofibrils reduced force proportional to [BLEB]. Two-photon Indo1-AM ratio-metric confocal line-scan microscopy revealed no impact of BLEB on the cytosolic Ca(2+) transient. BLEB reduced contractile force in skinned trabeculae without affecting tension-dependent myofilament ATPase activity. We conclude that BLEB specifically uncouples cardiac myofilament activation from Ca(2+) activation without affecting EC coupling or cross-bridge cycling parameters. This agent could be useful to uncouple myofilament contractility from electrical events that lead to sarcoplasmic reticulum Ca(2+) release in the cardiac myocyte (uncoupling agent) However, the compound is very sensitive to light, a property that limits its application to mechanistic physiological studies.

Research paper thumbnail of Reply to Smith letter: Controversy persists after over 100 years of the Frank–Starling mechanism

Journal of Molecular and Cellular Cardiology, 2010

Research paper thumbnail of Troponin phosphorylation and myofilament Ca2+-sensitivity in heart failure: Increased or decreased?

Journal of Molecular and Cellular Cardiology, 2008

Heart failure is characterised by depressed myocyte contractility and is considered to involve a ... more Heart failure is characterised by depressed myocyte contractility and is considered to involve a complex malfunction of adrenergic regulation, Ca 2+ -handling and the contractile apparatus. Most studies on the contractile apparatus have focused on troponin, the Ca 2+ -dependent regulator of myofibrillar activity. Importantly, phosphorylation of troponin I secondary to beta-adrenergic receptor activation is known to induce reduced myofilament Ca 2+ sensitivity. In muscle samples from explanted failing human hearts, troponin I phosphorylation levels are very low and Ca 2+sensitivity is high. In contrast, some animal models used to study the mechanisms of heart failure give the opposite result-high levels of troponin I phosphorylation and low Ca 2+ -sensitivity. Which is right?

Research paper thumbnail of Cardiac actin capping protein is an essential element in protein kinase C signaling to the myofilament

Journal of Molecular and Cellular Cardiology, 2001

Research paper thumbnail of Myofilament length dependent activation

Journal of Molecular and Cellular Cardiology, 2010

The Frank-Starling law of the heart describes the interrelationship between end-diastolic volume ... more 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 Ca 2+ 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.

Research paper thumbnail of Control of cardiac myofilament activation and PKC-βII signaling through the actin capping protein, CapZ

Journal of Molecular and Cellular Cardiology, 2006

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilame... more Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.

Research paper thumbnail of Increased Cross-bridge Cycling Kinetics after Exchange of C-terminal Truncated Troponin I in Skinned Rat Cardiac Muscle

Journal of Biological Chemistry, 2008

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not full... more The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.

Research paper thumbnail of The Troponin C G159D Mutation Blunts Myofilament Desensitization Induced by Troponin I Ser23/24 Phosphorylation

Circulation Research, 2007

Striated muscle contraction is regulated by the binding of Ca 2ϩ to the N-terminal regulatory lob... more Striated muscle contraction is regulated by the binding of Ca 2ϩ to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, ␤-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca 2ϩ -dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca 2ϩ in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca 2ϩ sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca 2ϩ -sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca 2ϩ -binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca 2ϩ -binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through ␤-adrenergic signaling as well as the development of cardiomyopathy.

Research paper thumbnail of Intracellular Localization and Functional Effects of P21-Activated Kinase-1 (Pak1) in Cardiac Myocytes

Circulation Research, 2004

Research paper thumbnail of Restoration of Resting Sarcomere Length After Uniaxial Static Strain Is Regulated by Protein Kinase C  and Focal Adhesion Kinase

Circulation Research, 2004

Physiological or pathological stresses and strains produce longer or wider muscle cells, but rest... more Physiological or pathological stresses and strains produce longer or wider muscle cells, but resting sarcomere length remains constant. Our goal was to investigate the cellular mechanisms for controlling this optimal, resting sarcomere length. To do so, we cultured neonatal rat cardiomyocytes on microfabricated peg-and-groove, laminincoated silicone surfaces and applied a uniaxial static strain of 10%. Sarcomere length was accurately measured by fast Fourier transform analysis of images before, within 5 minutes of, and 4 to 6 hours after imposition of the strain. Sarcomere length of aligned cardiomyocytes (1.94Ϯ0.07 m) was lengthened acutely (2.06Ϯ0.06 m), and recovered (1.95Ϯ0.07 m) by 4 hours. Puromycin, an mRNA translational inhibitor, prevented recovery of resting sarcomere length by 4 hours, thus indicating a requirement for new protein synthesis in the recovery process. Furthermore, activation of protein kinase C⑀ (PKC⑀) was necessary for length recovery, as nonselective PKC inhibitors [staurosporine (5 mol/L) and chelerythrine chloride (10 mol/L)], and a replication-defective adenovirus (Adv) encoding a dominant-negative mutant of PKC⑀ prevented the restoration of sarcomere length. To assess the importance of focal adhesion complexes, cardiomyocytes were infected with an Adv encoding a dominant-negative inhibitor of focal adhesion kinase (FAK) (Adv-GFP-FRNK). Adv-GFP-FRNK also prevented resting sarcomere length recovery, whereas a control Adv encoding only GFP did not. In conclusion, using our novel culture system, we provide evidence indicating that the length remodeling process requires new protein synthesis, PKC⑀ and FAK. (Circ Res. 2004;94:642-649.)

Research paper thumbnail of Actin Capping Protein: An Essential Element in Protein Kinase Signaling to the Myofilaments

Circulation Research, 2002

Research paper thumbnail of Titin Isoform Switch in Ischemic Human Heart Disease

Circulation, 2002

Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic... more Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic pressure, which follows from increased myocardial stiffness resulting from upregulated collagen expression. In addition to collagen, a main determinant of stiffness is titin, whose role in ischemia-induced left ventricular stiffening was studied here. Human heart sarcomeres coexpress 2 principal titin isoforms, a more compliant N2BA isoform and a stiffer N2B isoform. In comparison, normal rat hearts express almost no N2BA titin. Gel electrophoresis and immunoblotting were used to determine the N2BA-to-N2B titin isoform ratio in nonischemic human hearts and nonnecrotic left ventricle of coronary artery disease (CAD) patients. The average N2BA-to-N2B ratio was 47:53 in severely diseased CAD transplanted hearts and 32:68 in nonischemic transplants. In normal donor hearts and donor hearts with CAD background, relative N2BA titin content was approximately 30%. The titin isoform shift in CAD transplant hearts coincided with a high degree of modifications of cardiac troponin I, probably indicating increased preload. Immunofluorescence microscopy on CAD transplant specimens showed a regular cross-striated arrangement of titin and increased expression of collagen and desmin. Force measurements on isolated myofibrils revealed reduced passive-tension levels in sarcomeres of CAD hearts with high left ventricular end-diastolic pressure compared with sarcomeres of normal hearts. In a rat model of ischemia-induced myocardial infarction (left anterior descending coronary artery ligature), 43% of animals, but only 14% of sham-operated animals, showed a distinct N2BA titin band on gels. A titin isoform switch was observed in chronically ischemic human hearts showing extensive remodeling, which necessitated cardiac transplantation. The shift, also confirmed in rat hearts, caused reduced titin-derived myofibrillar stiffness. Titin modifications in long-term ischemic myocardium could impair the ability of the heart to use the Frank-Starling mechanism.

Research paper thumbnail of Impact Of N-terminal Truncation Of Cardiac Troponin I On Myofilament Chemo-mechanical Transduction: Implications For The Enhanced Cardiac Function In Hemodynamic Adaptation

Biophysical Journal, 2009

To address this issue, a selective exchange procedure was used in which 50% and 70% of the endoge... more To address this issue, a selective exchange procedure was used in which 50% and 70% of the endogenous cTn complex in permeabilized human cardiomyocytes was exchanged with recombinant unphosphorylated human cTn. Cardiomyocytes isolated from healthy donor hearts showed almost saturated phosphorylation levels at the ser23/24 of cTnI. Endogenous phosphorylated cTn of donor cardiomyocytes (pCa 50 ¼ 5.4550.03) was exchanged with 0.5 and 1.0 mg/ml unphosphorylated recombinant human cTn (cTn-U), which resulted in an increase in Ca 2þ -sensitivity (DpCa 50 ¼0.08). Subsequent incubation of the cells with PKA reversed Ca 2þ -sensitivity to baseline levels (pCa 50 ¼ 5.4650.03).

Research paper thumbnail of Cardiac Troponin I Threonine 144 phosphorylation: impact on myofilament function

Biophysical Journal, 2009

It is well known that protein kinase A (PKA) decreases Ca 2þ sensitivity in cardiac muscle via ph... more It is well known that protein kinase A (PKA) decreases Ca 2þ sensitivity in cardiac muscle via phosphorylation of troponin I (TnI). In the present study, we directly tested whether PKA-based phosphorylation of cardiac TnI universally modulates Ca 2þ sensitivity regardless of the type of muscle, by taking advantage of our Tn exchange technique (Terui et al., J Gen. Physiol. 131;275-283:2008). Troponins were extracted from porcine ventricular and rabbit fast skeletal muscles (Ca 2þ sensitivity: former < latter). Without Tn exchange, PKA decreased Ca 2þ sensitivity in cardiac (porcine ventricular) muscle, associated with enhanced phosphorylation of TnI. Reconstitution of cardiac muscle with the skeletal Tn complex (sTn) not only increased Ca 2þ sensitivity but abolished the PKA effect, suggesting that phosphorylation of TnI, but not of myosin-binding protein C, is primarily responsible for the PKA-based reduction in Ca 2þ sensitivity. Reconstitution of rabbit psoas muscle with the cardiac Tn complex (cTn) decreased Ca 2þ sensitivity, as previously reported by us (Terui et al., J Gen. Physiol. 131;275-283:2008). PKA decreased Ca 2þ sensitivity in cTn-reconstituted skeletal muscle, and subsequent exchange for sTn restored Ca 2þ sensitivity to the original level. A similar result was obtained when skeletal muscle was reconstituted with the hybrid Tn complex (i.e., cTnI-cTnC-sTnT), suggesting that the troponin I-C complex, but not TnT, is essential for PKA-based modulation of Ca 2þ sensitivity. These findings support the notion that PKA-based phosphorylation of TnI universally modulates Ca 2þ sensitivity regardless of the type of muscle. Threonine 144 (T144) in the inhibitory region of cardiac troponin I (cTnI) is an important site for PKC mediated phosphorylation in the heart. In addition, presence of this residue is sufficient to impart length dependent activation (LDA) properties onto the cardiac sarcomere (Tachampa Circ. Res., 2008). Here we studied the functional impact of a charge mutation so as to mimic phosphorylation of this residue (T144E). Wild-type (WT) or cTnI-T144E containing recombinant troponin (cTn) complexes were exchanged for endogenous cTn in skinned rat cardiac trabeculae. Force and ATPase activity were measured as function of [Ca2þ] at short (2.0 mm) or long (2.2 mm) sarcomere length. T144E induced decreased maximum force development (Fmax) and Calcium sensitivity (EC50), increased cross-bridge cycling rate (tension cost) but, in contrast, did not affect LDA (DEC50). We conclude that T144E affects cross-bridge cycling and recruitment independent of sarcomere length.

Research paper thumbnail of Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

Pflügers Archiv - European Journal of Physiology, 2008

The present study examined the contribution of myofilament contractile proteins to regional funct... more The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca 2+ sensitivity, and cross-bridge cycling kinetics (K tr ) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca 2+ sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K tr was unchanged by stretch, but was higher in EPI cells possibly due to a higher α-MHC content. Submaximal Ca 2+ -activated K tr increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosinbinding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.

Research paper thumbnail of Beneficial effects of SR33805 in failing myocardium

Cardiovascular Research, 2011

1 This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca 2+... more 1 This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca 2+antagonistic properties, on the contractile properties of intact and skinned rat ventricular myocytes. 2 On intact cells loaded with the Ca 2+ -fluorescent indicator Indo-1, the application of low concentrations of SR33805 enhanced the amplitude of unloaded cell shortening and decreased the duration of cell shortening. Amplitude of the Ca 2+ transient was also decreased. 3 These effects were accompanied with a shortening of the action potential and a dose-dependent blockade of L-type calcium current (IC 50 ¼ 2.4 Â 10 À8 M). 4 On skinned cardiac cells, the application of a low SR33805 concentration (10 À8 M) induced a significant increase in maximal Ca 2+ -activated force at the two-tested sarcomere lengths (SLs), 1.9 and 2.3 mm. 5 The application of a larger dose of SR33805 (10 À6 -10 À5 M) induced a significant leftward shift of the tension -pCa relation that accounts for Ca 2+ -sensitization of the myofilaments, particularly at 2.3 mm SL. 6 In conclusion, despite its strong Ca 2+ -antagonistic properties SR33805 increases cardiac cell contractile activity as a consequence of its Ca 2+ -sensitizing effects. These effects are attributable to both an increase in the maximal Ca 2+ -activated force and a length-dependent Ca 2+ -sensitization.

Research paper thumbnail of Activation Cardiac Troponin I Threonine 144: Role in Myofilament LengthDependent

Myofilament length-dependent activation is the main cellular mechanism responsible for the Frank-... more Myofilament length-dependent activation is the main cellular mechanism responsible for the Frank-Starling law of the heart. All striated muscle display lengthdependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length-dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length-dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnI Thr>Pro and ssTnI Pro>Thr . Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force-Ca 2؉ relationships were determined to derive myofilament Ca 2؉ sensitivity (EC 50 ) at 2 sarcomere lengths: 2.0 and 2.2 m (n‫.)7؍‬ Myofilament lengthdependent activation was indexed as ⌬EC 50 , the difference in EC 50 between sarcomere lengths of 2.0 and 2.2 m. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced ⌬EC 50 from 1.26؎0.30 to 0.19؎0.04 mol/L. A similar reduction also could be observed when Tn contained cTnI Thr>Pro (⌬EC 50 ‫40.0؎42.0؍‬ mol/L), whereas the presence of ssTnI Pro>Thr increased ⌬EC 50 to 0.94؎0.12 mol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length-dependent activation.

Research paper thumbnail of cGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation: A Tale of Two Isoforms

Research paper thumbnail of Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing

The Journal of Physiology, 2003

Research paper thumbnail of Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

Pflügers Archiv - European Journal of Physiology, 2008

The present study examined the contribution of myofilament contractile proteins to regional funct... more The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca 2+ sensitivity, and cross-bridge cycling kinetics (K tr ) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca 2+ sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K tr was unchanged by stretch, but was higher in EPI cells possibly due to a higher α-MHC content. Submaximal Ca 2+ -activated K tr increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosinbinding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.

Research paper thumbnail of Blebbistatin: use as inhibitor of muscle contraction

Pflügers Archiv - European Journal of Physiology, 2008

Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In... more Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In this study, we tested BLEB in intact and skinned isolated rat cardiac trabeculae, rat intact myocytes, and single rabbit psoas myofibrils. BLEB (10 muM) reduced twitch force and cell shortening that was reversed by exposure to light. BLEB treatment of skinned trabeculae in the dark (1 hr) reduced Ca(2+)-activated force (EC(50) = 0.38 +/- 0.03 muM). Rapid (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5 ms) BLEB application in Ca(2+)-activated rabbit myofibrils reduced force proportional to [BLEB]. Two-photon Indo1-AM ratio-metric confocal line-scan microscopy revealed no impact of BLEB on the cytosolic Ca(2+) transient. BLEB reduced contractile force in skinned trabeculae without affecting tension-dependent myofilament ATPase activity. We conclude that BLEB specifically uncouples cardiac myofilament activation from Ca(2+) activation without affecting EC coupling or cross-bridge cycling parameters. This agent could be useful to uncouple myofilament contractility from electrical events that lead to sarcoplasmic reticulum Ca(2+) release in the cardiac myocyte (uncoupling agent) However, the compound is very sensitive to light, a property that limits its application to mechanistic physiological studies.

Research paper thumbnail of Reply to Smith letter: Controversy persists after over 100 years of the Frank–Starling mechanism

Journal of Molecular and Cellular Cardiology, 2010

Research paper thumbnail of Troponin phosphorylation and myofilament Ca2+-sensitivity in heart failure: Increased or decreased?

Journal of Molecular and Cellular Cardiology, 2008

Heart failure is characterised by depressed myocyte contractility and is considered to involve a ... more Heart failure is characterised by depressed myocyte contractility and is considered to involve a complex malfunction of adrenergic regulation, Ca 2+ -handling and the contractile apparatus. Most studies on the contractile apparatus have focused on troponin, the Ca 2+ -dependent regulator of myofibrillar activity. Importantly, phosphorylation of troponin I secondary to beta-adrenergic receptor activation is known to induce reduced myofilament Ca 2+ sensitivity. In muscle samples from explanted failing human hearts, troponin I phosphorylation levels are very low and Ca 2+sensitivity is high. In contrast, some animal models used to study the mechanisms of heart failure give the opposite result-high levels of troponin I phosphorylation and low Ca 2+ -sensitivity. Which is right?

Research paper thumbnail of Cardiac actin capping protein is an essential element in protein kinase C signaling to the myofilament

Journal of Molecular and Cellular Cardiology, 2001

Research paper thumbnail of Myofilament length dependent activation

Journal of Molecular and Cellular Cardiology, 2010

The Frank-Starling law of the heart describes the interrelationship between end-diastolic volume ... more 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 Ca 2+ 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.

Research paper thumbnail of Control of cardiac myofilament activation and PKC-βII signaling through the actin capping protein, CapZ

Journal of Molecular and Cellular Cardiology, 2006

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilame... more Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.

Research paper thumbnail of Increased Cross-bridge Cycling Kinetics after Exchange of C-terminal Truncated Troponin I in Skinned Rat Cardiac Muscle

Journal of Biological Chemistry, 2008

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not full... more The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.

Research paper thumbnail of The Troponin C G159D Mutation Blunts Myofilament Desensitization Induced by Troponin I Ser23/24 Phosphorylation

Circulation Research, 2007

Striated muscle contraction is regulated by the binding of Ca 2ϩ to the N-terminal regulatory lob... more Striated muscle contraction is regulated by the binding of Ca 2ϩ to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, ␤-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca 2ϩ -dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca 2ϩ in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca 2ϩ sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca 2ϩ -sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca 2ϩ -binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca 2ϩ -binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through ␤-adrenergic signaling as well as the development of cardiomyopathy.

Research paper thumbnail of Intracellular Localization and Functional Effects of P21-Activated Kinase-1 (Pak1) in Cardiac Myocytes

Circulation Research, 2004

Research paper thumbnail of Restoration of Resting Sarcomere Length After Uniaxial Static Strain Is Regulated by Protein Kinase C  and Focal Adhesion Kinase

Circulation Research, 2004

Physiological or pathological stresses and strains produce longer or wider muscle cells, but rest... more Physiological or pathological stresses and strains produce longer or wider muscle cells, but resting sarcomere length remains constant. Our goal was to investigate the cellular mechanisms for controlling this optimal, resting sarcomere length. To do so, we cultured neonatal rat cardiomyocytes on microfabricated peg-and-groove, laminincoated silicone surfaces and applied a uniaxial static strain of 10%. Sarcomere length was accurately measured by fast Fourier transform analysis of images before, within 5 minutes of, and 4 to 6 hours after imposition of the strain. Sarcomere length of aligned cardiomyocytes (1.94Ϯ0.07 m) was lengthened acutely (2.06Ϯ0.06 m), and recovered (1.95Ϯ0.07 m) by 4 hours. Puromycin, an mRNA translational inhibitor, prevented recovery of resting sarcomere length by 4 hours, thus indicating a requirement for new protein synthesis in the recovery process. Furthermore, activation of protein kinase C⑀ (PKC⑀) was necessary for length recovery, as nonselective PKC inhibitors [staurosporine (5 mol/L) and chelerythrine chloride (10 mol/L)], and a replication-defective adenovirus (Adv) encoding a dominant-negative mutant of PKC⑀ prevented the restoration of sarcomere length. To assess the importance of focal adhesion complexes, cardiomyocytes were infected with an Adv encoding a dominant-negative inhibitor of focal adhesion kinase (FAK) (Adv-GFP-FRNK). Adv-GFP-FRNK also prevented resting sarcomere length recovery, whereas a control Adv encoding only GFP did not. In conclusion, using our novel culture system, we provide evidence indicating that the length remodeling process requires new protein synthesis, PKC⑀ and FAK. (Circ Res. 2004;94:642-649.)

Research paper thumbnail of Actin Capping Protein: An Essential Element in Protein Kinase Signaling to the Myofilaments

Circulation Research, 2002

Research paper thumbnail of Titin Isoform Switch in Ischemic Human Heart Disease

Circulation, 2002

Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic... more Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic pressure, which follows from increased myocardial stiffness resulting from upregulated collagen expression. In addition to collagen, a main determinant of stiffness is titin, whose role in ischemia-induced left ventricular stiffening was studied here. Human heart sarcomeres coexpress 2 principal titin isoforms, a more compliant N2BA isoform and a stiffer N2B isoform. In comparison, normal rat hearts express almost no N2BA titin. Gel electrophoresis and immunoblotting were used to determine the N2BA-to-N2B titin isoform ratio in nonischemic human hearts and nonnecrotic left ventricle of coronary artery disease (CAD) patients. The average N2BA-to-N2B ratio was 47:53 in severely diseased CAD transplanted hearts and 32:68 in nonischemic transplants. In normal donor hearts and donor hearts with CAD background, relative N2BA titin content was approximately 30%. The titin isoform shift in CAD transplant hearts coincided with a high degree of modifications of cardiac troponin I, probably indicating increased preload. Immunofluorescence microscopy on CAD transplant specimens showed a regular cross-striated arrangement of titin and increased expression of collagen and desmin. Force measurements on isolated myofibrils revealed reduced passive-tension levels in sarcomeres of CAD hearts with high left ventricular end-diastolic pressure compared with sarcomeres of normal hearts. In a rat model of ischemia-induced myocardial infarction (left anterior descending coronary artery ligature), 43% of animals, but only 14% of sham-operated animals, showed a distinct N2BA titin band on gels. A titin isoform switch was observed in chronically ischemic human hearts showing extensive remodeling, which necessitated cardiac transplantation. The shift, also confirmed in rat hearts, caused reduced titin-derived myofibrillar stiffness. Titin modifications in long-term ischemic myocardium could impair the ability of the heart to use the Frank-Starling mechanism.

Research paper thumbnail of Impact Of N-terminal Truncation Of Cardiac Troponin I On Myofilament Chemo-mechanical Transduction: Implications For The Enhanced Cardiac Function In Hemodynamic Adaptation

Biophysical Journal, 2009

To address this issue, a selective exchange procedure was used in which 50% and 70% of the endoge... more To address this issue, a selective exchange procedure was used in which 50% and 70% of the endogenous cTn complex in permeabilized human cardiomyocytes was exchanged with recombinant unphosphorylated human cTn. Cardiomyocytes isolated from healthy donor hearts showed almost saturated phosphorylation levels at the ser23/24 of cTnI. Endogenous phosphorylated cTn of donor cardiomyocytes (pCa 50 ¼ 5.4550.03) was exchanged with 0.5 and 1.0 mg/ml unphosphorylated recombinant human cTn (cTn-U), which resulted in an increase in Ca 2þ -sensitivity (DpCa 50 ¼0.08). Subsequent incubation of the cells with PKA reversed Ca 2þ -sensitivity to baseline levels (pCa 50 ¼ 5.4650.03).

Research paper thumbnail of Cardiac Troponin I Threonine 144 phosphorylation: impact on myofilament function

Biophysical Journal, 2009

It is well known that protein kinase A (PKA) decreases Ca 2þ sensitivity in cardiac muscle via ph... more It is well known that protein kinase A (PKA) decreases Ca 2þ sensitivity in cardiac muscle via phosphorylation of troponin I (TnI). In the present study, we directly tested whether PKA-based phosphorylation of cardiac TnI universally modulates Ca 2þ sensitivity regardless of the type of muscle, by taking advantage of our Tn exchange technique (Terui et al., J Gen. Physiol. 131;275-283:2008). Troponins were extracted from porcine ventricular and rabbit fast skeletal muscles (Ca 2þ sensitivity: former < latter). Without Tn exchange, PKA decreased Ca 2þ sensitivity in cardiac (porcine ventricular) muscle, associated with enhanced phosphorylation of TnI. Reconstitution of cardiac muscle with the skeletal Tn complex (sTn) not only increased Ca 2þ sensitivity but abolished the PKA effect, suggesting that phosphorylation of TnI, but not of myosin-binding protein C, is primarily responsible for the PKA-based reduction in Ca 2þ sensitivity. Reconstitution of rabbit psoas muscle with the cardiac Tn complex (cTn) decreased Ca 2þ sensitivity, as previously reported by us (Terui et al., J Gen. Physiol. 131;275-283:2008). PKA decreased Ca 2þ sensitivity in cTn-reconstituted skeletal muscle, and subsequent exchange for sTn restored Ca 2þ sensitivity to the original level. A similar result was obtained when skeletal muscle was reconstituted with the hybrid Tn complex (i.e., cTnI-cTnC-sTnT), suggesting that the troponin I-C complex, but not TnT, is essential for PKA-based modulation of Ca 2þ sensitivity. These findings support the notion that PKA-based phosphorylation of TnI universally modulates Ca 2þ sensitivity regardless of the type of muscle. Threonine 144 (T144) in the inhibitory region of cardiac troponin I (cTnI) is an important site for PKC mediated phosphorylation in the heart. In addition, presence of this residue is sufficient to impart length dependent activation (LDA) properties onto the cardiac sarcomere (Tachampa Circ. Res., 2008). Here we studied the functional impact of a charge mutation so as to mimic phosphorylation of this residue (T144E). Wild-type (WT) or cTnI-T144E containing recombinant troponin (cTn) complexes were exchanged for endogenous cTn in skinned rat cardiac trabeculae. Force and ATPase activity were measured as function of [Ca2þ] at short (2.0 mm) or long (2.2 mm) sarcomere length. T144E induced decreased maximum force development (Fmax) and Calcium sensitivity (EC50), increased cross-bridge cycling rate (tension cost) but, in contrast, did not affect LDA (DEC50). We conclude that T144E affects cross-bridge cycling and recruitment independent of sarcomere length.