Chee Chew Lim

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 μmol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in ∼3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.

Heterozygous Cellular Glutathione Peroxidase Deficiency in the Mouse Abnormalities in Vascular and Cardiac Function and Structure

Background—Oxidant stress has been implicated in the pathogenesis of atherothrombosis and other vascular disorders accompanied by endothelial dysfunction. Glutathione peroxidases (GPx) play an important role in the cellular defense against oxidant stress by utilizing glutathione (GSH) to reduce lipid hydroperoxides and hydrogen peroxide to their corresponding alcohols. Cellular GPx (GPx-1) is the principal intracellular isoform of GPx. We hypothesized that GPx-1 deficiency per se induces endothelial dysfunction and structural vascular abnormalities through increased oxidant stress. Methods and Results—A murine model of heterozygous deficiency of GPx-1 (GPx+/−) was investigated to examine this hypothesis. Mesenteric arterioles in GPx-1+/− mice demonstrated vasoconstriction to acetylcholine compared with vasodilation in wild-type mice (maximal change in vessel diameter, −13.0±2.8% versus 13.2±2.8%, P <0.0001). We also noted an increase in the plasma and aortic levels of the isoprostane iPF2&agr;-III, a marker of oxidant stress, in GPx-1+/− mice compared with wild-type mice (170.4±23 pg/mL plasma versus 98.7±7.1 pg/mL plasma, P <0.03; 11.7±0.87 pg/mg aortic tissue versus 8.2±0.55 pg/mg aortic tissue, P <0.01). Histological sections from the coronary vasculature of GPx-1+/− mice show increased perivascular matrix deposition, an increase in the number of adventitial fibroblasts, and intimal thickening. These structural abnormalities in the myocardial vasculature were accompanied by diastolic dysfunction after ischemia-reperfusion. Conclusions—These findings demonstrate that heterozygous deficiency of GPx-1 leads to endothelial dysfunction, possibly associated with increased oxidant stress, and to significant structural vascular and cardiac abnormalities. These data illustrate the importance of this key antioxidant enzyme in functional and structural responses of the mammalian cardiovascular system.

Glucose-6-Phosphate Dehydrogenase Modulates Cytosolic Redox Status and Contractile Phenotype in Adult Cardiomyocytes

&NA; —Reactive oxygen species (ROS)‐mediated cell injury contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. Protection against ROS requires maintenance of endogenous thiol pools, most importantly, reduced glutathione (GSH), by NADPH. In cardiomyocytes, GSH resides in two separate cellular compartments: the mitochondria and cytosol. Although mitochondrial GSH is maintained largely by transhydrogenase and isocitrate dehydrogenase, the mechanisms responsible for sustaining cytosolic GSH remain unclear. Glucose‐6‐phosphate dehydrogenase (G6PD) functions as the first and rate‐limiting enzyme in the pentose phosphate pathway, responsible for the generation of NADPH in a reaction coupled to the de novo production of cellular ribose. We hypothesized that G6PD is required to maintain cytosolic GSH levels and protect against ROS injury in cardiomyocytes. We found that in adult cardiomyocytes, G6PD activity is rapidly increased in response to cellular oxidative stress, with translocation of G6PD to the cell membrane. Furthermore, inhibition of G6PD depletes cytosolic GSH levels and subsequently results in cardiomyocyte contractile dysfunction through dysregulation of calcium homeostasis. Cardiomyocyte dysfunction was reversed through treatment with either a thiol‐repleting agent (L‐2‐oxothiazolidine‐4‐carboxylic acid) or antioxidant treatment (Eukarion‐134), but not with exogenous ribose. Finally, in a murine model of G6PD deficiency, we demonstrate the development of in vivo adverse structural remodeling and impaired contractile function over time. We, therefore, conclude that G6PD is a critical cytosolic antioxidant enzyme, essential for maintenance of cytosolic redox status in adult cardiomyocytes. Deficiency of G6PD may contribute to cardiac dysfunction through increased susceptibility to free radical injury and impairment of intracellular calcium transport. The full text of this article is available online at http://www.circresaha.org. (Circ Res. 2003;93:e9‐e16.)

Titin Determines the Frank-Starling Relation in Early Diastole

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.

Redox-mediated reciprocal regulation of SERCA and Na+-Ca2+ exchanger contributes to sarcoplasmic reticulum Ca2+ depletion in cardiac myocytes.

Myocardial failure is associated with increased oxidative stress and abnormal excitation-contraction coupling characterized by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores and a reduction in Ca(2+)-transient amplitude. Little is known about the mechanisms whereby oxidative stress affects Ca(2+) handling and contractile function; however, reactive thiols may be involved. We used an in vitro cardiomyocyte system to test the hypothesis that short-term oxidative stress induces SR Ca(2+) depletion via redox-mediated regulation of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) and the sodium-Ca(2+) exchanger (NCX) and that this is associated with thiol oxidation. Adult rat ventricular myocytes paced at 5 Hz were superfused with H(2)O(2) (100 microM, 15 min). H(2)O(2) caused a progressive decrease in cell shortening followed by diastolic arrest, which was associated with decreases in SR Ca(2+) content, systolic [Ca(2+)](i), and Ca(2+)-transient amplitude, but no change in diastolic [Ca(2+)](i). H(2)O(2) caused reciprocal effects on the activities of SERCA (decreased) and NCX (increased). Pretreatment with the NCX inhibitor KB-R7943 before H(2)O(2) increased diastolic [Ca(2+)](i) and mimicked the effect of SERCA inhibition with thapsigargin. These functional effects were associated with oxidative modification of thiols on both SERCA and NCX. In conclusion, redox-mediated SR Ca(2+) depletion involves reciprocal regulation of SERCA and NCX, possibly via direct oxidative modification of both proteins.

Impaired lusitropy-frequency in the aging mouse : role of Ca2+-handling proteins and effects of isoproterenol

We examined the relationship between age-associated lusitropic impairment, heart rate, and Ca2+-handling proteins and assessed the efficacy of increasing left ventricular (LV) relaxation via β-adrenergic stimulation in adult and aging mouse hearts. LV function was measured in isolated, isovolumic blood-perfused hearts from adult (5 mo), old (24 mo), and senescent (34 mo) mice. Hearts were paced from 5 to 10 Hz, returned to 7 Hz, exposed to 10-6 M isoproterenol, and paced again from 7 to 10 Hz. Age-related alterations in Na+/Ca2+exchanger (NCX), sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), and phospholamban (PLB) levels were assessed by immunoblot. Despite preserved contractile performance, aging caused impaired lusitropy. Increased pacing caused an elevation in end-diastolic pressure that progressively worsened with age. The time constant of isovolumic pressure decay (τ) was significantly prolonged in old and senescent hearts compared with adults. Relative to adult hearts, the SERCA2a-to-PLB ratios were reduced 68 and 69%, and NCX were reduced 37 and 58% in old and senescent hearts, respectively. Isoproterenol completely reversed the age-associated lusitropic impairments. These data suggest that impaired lusitropy in aging mouse hearts is related to a decreased rate of cytosolic Ca2+ removal and that accelerating SR Ca2+ resequestration via β-adrenergic stimulation can reverse this impairment.

Cellular force measurements using single-spaced polymeric microstructures: isolating cells from base substrate

Mechanical force is one of the most important parameters in cellular physiological behavior. To quantify the cellular force locally and more precisely, soft material probes, such as bulk polymeric surfaces or raised individual polymeric structures, have been developed which are deformable by the cell. The extent of deformation and the elastic properties of the probes allow for calculation of the mechanical forces exerted by the cell. Bulk polymeric surfaces have the disadvantage of requiring computational intensive calculations due to the continuous distortion of a large area, and investigators have attempted to address this problem by using raised polymeric structures to simplify the derivation of cellular mechanical force. These studies, however, have ignored the possibility of formation of local adhesions of the cell to the underlying base substrate, which could result in inaccurate cellular force measurements. Clearly, there is a need to develop polymeric structures that can efficiently isolate the cells from the underlying base substrate, in order to eliminate the continuous distortion problem. In this paper, we demonstrate the measurement of cellular force in isolated cardiac myocytes using single-spaced polymeric microstructures. Each structure is 2 ?m in diameter and single-spaced packed. This geometry of the structures successfully isolates the cells from the underlying substrate. Displacement of the structures was measured in areas underneath the attached cell and at areas in close proximity to the cell. The results show that the individual structures underneath the cell were significantly displaced whereas no substantial strain in the underlying base substrate was detected. The mechanical force of the cell was derived from the displacements of individual structures upon multiplication with the locally determined spring constant. The force distribution reveals a parallel alignment as well as a periodic motion of the contractile units of the myocyte. The flexible fabrication methodology of the polymeric substrate and straightforward determination of minute forces provide a useful way to study cellular mechanical force.

Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes

Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia‐induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol‐3‐kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal‐related kinase (Erk) and c‐jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL‐positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro‐apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia‐induced cardiomyopathy.

Modulation of cardiac function: titin springs into action.

Sympathetic stimulation has become a central tenet in our understanding of how cardiac contractility is dynamically altered to accommodate the changing demands of the organism. The mechanisms by which acute and chronic sympathetic stimulation of the heart modulates cardiac output remain incompletely

Oriented and Vectorial Patterning of Cardiac Myocytes Using a Microfluidic Dielectrophoresis Chip - Towards Engineered Cardiac Tissue with Controlled Macroscopic Anisotropy

Recently, the ability to create engineered heart tissues with a preferential cell orientation has gained much interest. Here, we present a novel method to construct a cardiac myocyte tissue-like structure using a combination of dielectrophoresis and electro-orientation via a microfluidic chip. The device includes a top home-made silicone chamber containing microfluidic channels and bottom integrated microelectrodes which are patterned on a glass slide to generate dielectrophoresis force and orientation torque. Using the interdigitated-castellated microelectrodes, the induction of a mutually attractive dielectrophoretic force between cardiac myocytes can lead to cells moving close to each other and forming a tissue-like structure with orientation along the alternating current (ac) electric field between the microelectrode gaps. Both experiments and analysis indicate that a large orientation torque and force can be achieved by choosing an optimal frequency around 2 MHz and decreasing the conductivity of medium to a relatively low level. Finally, electromechanical experiments and biopolar impedance measurements were performed to demonstrate the structural and functional anisotropy of electro-oriented structure