Pieter P. de Tombe

Functional consequences of caspase activation in cardiac myocytes

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.

Titin Isoform Switch in Ischemic Human Heart Disease

Background—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. Methods and Results—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 ≈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. Conclusions—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.

Approximate Model of Cooperative Activation and Crossbridge Cycling in Cardiac Muscle Using Ordinary Differential Equations

We develop a point model of the cardiac myofilament (MF) to simulate a wide variety of experimental muscle characterizations including Force-Ca relations and twitches under isometric, isosarcometric, isotonic, and auxotonic conditions. Complex MF behaviors are difficult to model because spatial interactions cannot be directly implemented as ordinary differential equations. We therefore allow phenomenological approximations with careful consideration to the relationships with the underlying biophysical mechanisms. We describe new formulations that avoid mean-field approximations found in most existing MF models. To increase the scope and applicability of the model, we include length- and temperature-dependent effects that play important roles in MF responses. We have also included a representation of passive restoring forces to simulate isolated cell shortening protocols. Possessing both computational efficiency and the ability to simulate a wide variety of muscle responses, the MF representation is well suited for coupling to existing cardiac cell models of electrophysiology and Ca-handling mechanisms. To illustrate this suitability, the MF model is coupled to the Chicago rabbit cardiomyocyte model. The combined model generates realistic appearing action potentials, intracellular Ca transients, and cell shortening signals. The combined model also demonstrates that the feedback effects of force on Ca binding to troponin can modify the cytosolic Ca transient.

Augmented Protein Kinase C-α–Induced Myofilament Protein Phosphorylation Contributes to Myofilament Dysfunction in Experimental Congestive Heart Failure

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-α may be of particular importance. Here, we examined PKC-α 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-α 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–[Ca2+] relation to obtain Ca2+-saturated maximal force (Fmax) and myofilament Ca2+ sensitivity (indexed by EC50) before and after incubation with PKC-α, protein phosphatase type 1 (PP1), or PP2a. PKC-α treatment induced a 30% decline in Fmax and 55% increase in the EC50 in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased Fmax (15%) and decreased EC50 (≈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-α. 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-α.

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

Abstract— We investigated intracellular localization and substrate specificity of P21-activated kinase-1 (Pak1) in rat cardiac myocytes. Pak1 is a serine/threonine protein kinase that is activated by Rac1/Cdc42 and important in signaling of stress responses. Yet the localization and in vivo function of Pak1 in heart cells is poorly understood. Studies reported here indicate that Pak1 physically interacts with protein phosphatase 2a and localizes to the Z-disk, cell membrane, intercalated disc, and nuclear membrane of adult rat heart myocytes. We compared levels of phosphorylation of cardiac troponin I (cTnI) in control myocytes with phosphorylation of cTnI and myosin binding protein C (C-protein) in myocytes with increased Pak1 activity. The increase in activity was induced by infection of myocytes with a recombinant adenovirus (AdPak1) containing cDNA for a constitutively active Pak1. Control cells were infected with a virus (AdLacZ) containing LacZ. Basal levels of phosphorylation of cTnI and C-protein were relatively high in the myocytes infected with AdLacZ. However, phosphorylation of cTnI and C-protein in cells expressing constitutively active Pak1 was significantly reduced compared with those expressing LacZ. Measurement of Ca2+ tension relations in single myocytes demonstrated that this reduction in phosphorylation of cTnI and C-protein was associated with the predicted increase in sensitivity to Ca2+. Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.

The Troponin C G159D Mutation Blunts Myofilament Desensitization Induced by Troponin I Ser23/24 Phosphorylation

Striated muscle contraction is regulated by the binding of Ca2+ to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, &bgr;-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 Ca2+-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 Ca2+ in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca2+ sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca2+-sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca2+-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca2+-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through &bgr;-adrenergic signaling as well as the development of cardiomyopathy.

Length-dependent activation in three striated muscle types of the rat.

The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca2+ is termed length‐dependent activation. Length‐dependent activation is a property of all striated muscles, yet the relative extent of length‐dependent activation between skeletal muscle and cardiac muscle is unclear. Although length‐dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length‐dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length‐dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca2+‐force relationship was determined at sarcomere lengths (SL) of 1.95 μm, 2.10 μm and 2.25 μm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca2+ than CM. Length‐dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca2+‐force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X‐ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL‐LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length‐dependent activation differs among the three types of striated muscle and (2) these differences in the length‐dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations.

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to β1-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following β1-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in β1-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca2+ tension and Ca2+-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca2+-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca2+ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to β1-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.

Correlation Between Myofilament Response to Ca2+ and Altered Dynamics of Contraction and Relaxation in Transgenic Cardiac Cells That Express β-Tropomyosin

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

p38-MAPK Induced Dephosphorylation of α-Tropomyosin Is Associated With Depression of Myocardial Sarcomeric Tension and ATPase Activity

Our objective in work presented here was to understand the mechanisms by which activated p38&agr; MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38&agr; MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38&agr; MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of &agr;-tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38&agr; MAPK hearts. Our experiments also showed that p38&agr; MAPK colocalizes with &agr;-actinin at the Z-disc and complexes with protein phosphatases (PP2&agr;, PP2&bgr;). These data are the first to indicate that chronic activation of p38&agr; MAPK directly depresses sarcomeric function in association with decreased phosphorylation of &agr;-tropomyosin.