Cardiomyocyte Membrane Research Articles

Paradoxical Toxicity of Cardioplegic Compounds on Ischemic Cardiomyocyte Using Optimal Design Strategy

The aim of this study was to evaluate the effects of major components of cardioplegic solutions on myocardial tissue submitted to prolonged cold ischemia. Our methodology was based on the simultaneous testing in the same series of experiments of many compounds (19 in number), which were included in the composition of 20 established solutions. All the experiments were performed by a matricial-predefined protocol that allows the evaluation of the protective or toxic effects of each of these 19 compounds. Pig hearts were removed and left ventricular myocardiums were cut into 320 pieces. For each solution tested, 8 pieces of myocardial tissue were incubated at 4 degrees C for 24 hours and 8 other pieces were incubated for 72 hours. At the end of incubation period, tissue injury was assessed by measuring the leakage of myocardial enzymes(glutamic-oxaloacetic transaminase, lactate dehydrogenase, creatine phosphokinase) into the incubation medium. Initially, the effects of each solution were evaluated, and then a mathematical analysis was performed and the effects of each compound deduced. After the 24-hour incubation period, pyruvate (5 mmol/liter), polyethylene glycol (5 mmol/liter), Ala-Gln (20 mmol/liter), and reduced glutathione (3 mmol/liter) showed toxic effects, whereas ethanol (1%) and calcium chloride (2 mmol/liter) seemed to be protective. After 72 hours' incubation, similar data were obtained; dextran 70 (0.57 mmol/liter) was also found to be deleterious. The results revealed surprising myocardial toxicity (enzymatic release) from components included in cardioplegic solutions. Some components would induce metabolic activation during prolonged hypothermic ischemia, which may be inappropriated and which may perhaps exacerbate damages by increasing membrane ruptures. This concept confirms eventual discrepant effects of preservative compounds on cardiomyocyte membrane during deep hypothermia, according to the metabolic state of the cell.

Ankyrin-based cardiac arrhythmias: a new class of channelopathies due to loss of cellular targeting

This review addresses a new mechanism for arrhythmia due to abnormal cellular localization of membrane ion channels and transporters. The focus is on ankyrins, a family of proteins that localize diverse membrane ion channels and transporters, and recent evidence that mutations affecting functions of ankyrins result in cardiac arrhythmia. A loss-of-function mutation of ankyrin-B in humans and a null mutation in mice result in a dominantly-inherited fatal cardiac arrhythmia initially classified as type 4 long QT syndrome. Characterization of additional probands suggests ankyrin-B mutations cause a new cardiac arrhythmia syndrome associated with sinus node dysfunction that is distinct from long QT syndrome. Ankyrin-B mutation results in elevated calcium transients in cardiomyocytes accompanied by loss of cellular targeting of Na/K ATPase, Na/Ca exchanger, and InsP3 receptor (all ankyrin-binding proteins) to cardiomyocyte membrane domains. The principal voltage-gated Na channel in heart, Nav1.5, is directly associated with ankyrin-G, which is encoded by a distinct gene from ankyrin-B. Mutation of Nav1.5 causing loss of binding to ankyrin-G results in Brugada syndrome and loss of targeting of Nav1.5 to the cell surface of cardiomyocytes. Ankyrin-B and ankyrin-G are recently recognized constituents of the heart that target diverse ion channels/pumps/transporters to physiologic sites of action in cardiomyocytes. Mutations of ankyrin-B cause a newly defined cardiac arrhythmia syndrome associated with abnormal calcium homeostasis in a mouse model. Ankyrin-G associates with the principal voltage-gated Na channel in the heart, and loss of this interaction due to mutation of Nav1.5 results in Brugada syndrome.

Plasma‐membrane K ATP channel‐mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia

Our recent data demonstrate that activation of pmKATP channels polarizes the membrane of cardiomyocytes and reduces Na+/Ca2+ exchange-mediated Ca2+ overload. However, it is important that these findings be extended into contractile models of hypoxia/reoxygenation injury to further test the notion that pmKATP channel activation affords protection against contractile dysfunction and calcium overload. Single rat heart right ventricular myocytes were enzymatically isolated, and cell contractility and Ca2+ transients in field-stimulated myocytes were measured in a cellular model of metabolic inhibition and reoxygenation. Activation of pmKATP with P-1075 (5 microM) or inhibition of the Na+/Ca2+ exchanger with KB-R7943 (5 microM)reduced reoxygenation-induced diastolic Ca2+ overload and improved the rate and magnitude of posthypoxic contractile recovery during the first few minutes of reoxygenation. Moreover,diastolic Ca2+ overload and posthypoxic contractile dysfunction were aggravated in ventricular myocytes either subjected to specific blockade of pmKATP with HMR1098 (20 microM) or expressing the dominant-negative pmKATP construct Kir6.2(AAA) in the presence of P-1075. Our results suggest that a common mechanism, involving resting membrane potential-modulated increases in diastolic [Ca2+]i, is responsible for the development of contractile dysfunction during reoxygenation following metabolic inhibition. This novel and highly plausible cellular mechanism for pmKATP-mediated cardioprotection may have direct clinical relevance as evidenced by the following findings: a hypokalemic polarizing cardioplegia solution supplemented with the pmKATP opener P-1075 improved Ca2+ homeostasis and recovery of function compared with hyperkalemic depolarizing St. Thomas' cardioplegia following contractile arrest in single ventricular myocytes and working rat hearts. We therefore propose that activation of pmKATP channels improves posthypoxic cardiac function via reductions in abnormal diastolic Ca2+ homeostasis mediated by reverse-mode Na+/Ca2+ exchange.

Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/dystrophin-deficient cardiomyopathic mice

Remodeling of adherens junction, gap junction, and desmosomal proteins at the intercalated discs of cardiomyocytes in heart characterizes several animal models of cardiomyopathy, especially dilated cardiac myopathy (DCM). In this study, we show that the tight junction protein, claudin-5, is present in cardiac muscle and localizes to the lateral membranes of cardiomyocytes in normal mice. We further examined claudin-5 in utrophin/dystrophin-deficient (double knockout, dko) mice, a mouse model of muscular dystrophy with cardiomyopathy, and found that claudin-5 mRNA and protein levels are decreased in dko hearts as compared with normal. Intercalated disc cell junction proteins, and another tight junction protein, zonula occludens-1 (ZO-1), are not altered in the dko mouse. Ultrastructural data from dko hearts also shows that the lateral membranes of cardiomyocytes exhibit an abnormal wavy appearance. These data demonstrate that claudin-5 is specifically altered in dko hearts, suggesting that alterations of the lateral membranes of cardiomyocytes, rather than intercalated discs, are associated with cardiomyopathy in the dko mouse.

The Role of “Dormant” Mechanisms in Regulation of K/Na Balance in the Rat Cardiac Muscle Cell during Hypoxia

Using the electron probe microanalysis, potassium and sodium concentrations were measured in Wistar rat cardiac muscle cells. Hypoxic conditions were modeled according to the protocol of global ischemia in the absence of perfusion or via anoxic perfusion without glucose. It was shown that depending on the scheme of realization of hypoxia the change with time of the intracellular potassium and sodium balance had different character. Based on the obtained data, the sequence of activation of the hypoxia-specific mechanisms of ion transport on the cardiomyocyte membrane is considered.

Immunofluorescence revealed the presence of NHE-1 in the nuclear membranes of rat cardiomyocytes and isolated nuclei of human, rabbit, and rat aortic and liver tissues

Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes' NHE-1 may play a role in the modulation of intranuclear pH.

Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1.

Adenosine is an important paracrine hormone in the cardiovascular system. Adenosine flux across cardiomyocyte membranes occurs mainly via equilibrative nucleoside transporters (ENTs). The role of the ENTs in adenosine physiology is poorly understood, particularly in response to metabolic stress such as hypoxia. Therefore, we investigated the effects of chronic hypoxia on ENT1, the predominant ENT isoform in cardiomyocytes. HL-1 cells (immortalized murine cardiomyocytes) were exposed to hypoxia (2% O2) for 0-20 h. Cell viability, lactate dehydrogenase (LDH) release, glucose uptake, GLUT1 and GLUT4 protein, adenosine uptake, PKC activity, translocation profiles of PKCdelta and, nitrobenzylthioinosine (NBTI) binding and mENT1 mRNA levels were measured. The role of PKC in regulating mENT1 was further investigated using phorbol ester (100 nM, 18 h) and a dominant negative PKC construct, pSVK3PKC1-401. HL-1 cells have typical cardiomyocyte responses to hypoxia based on cell viability, LDH release, glucose uptake and GLUT protein levels. Hypoxia (8-20 h) down-regulates mENT1-dependent adenosine uptake, NBTI-binding and PKC but not PKCdelta in HL-1 cells. Abrogation of PKC activity using chronic phorbol ester or a dominant negative PKC mimicked the effect of hypoxia on adenosine uptake suggesting that PKC is involved in regulation of mENT1. Hypoxia (4 h) decreases mENT1 mRNA, which returns to basal levels by 20 h. Chronic hypoxia down-regulates mENT1 activity possibly via PKC. Hypoxia and PKC also regulate mENT1 RNA levels. Cardiomyocytes may regulate mENT1 (via PKC) to modulate release and/or uptake of adenosine. However, the relationship between mENT1 mRNA levels, protein levels and functional transport is complex.

ClC-3-independent, PKC-dependent Activity of Volume-sensitive Cl- Channel in Mouse Ventricular Cardiomyocytes

Volume-sensitive outwardly rectifying (VSOR) Cl^- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl^- channel in cardiomyocytes, the properties of VSOR Cl^- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3^-/-) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3^+/+) and heterozygous (Clcn3^+/-) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl^- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl^- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl^- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl^- currents. We thus conclude that functional expression of VSOR Cl^- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.

Increased calcium and decreased magnesium concentrations and an increased calcium/magnesium ratio in spontaneously hypertensive rats versus Wistar-Kyoto rats: relation to arteriosclerosis

Alterations in the metabolism of calcium and magnesium have been implicated in the pathogenesis of primary hypertension. Calcium influx across the external cellular membrane in smooth muscle cells and cardiomyocytes plays a crucial role in the control of cellular excitation contraction and impulse propagation. Intracellular calcium and magnesium concentrations are controlled by reversible binding to specific calcium-binding proteins. The calcium and magnesium flux across the external membrane is regulated by a calcium pump (calcium-magnesium-ATPase), calcium channels, and binding to the membrane. In cell membranes and in lymphocytes of essential hypertensives our group showed increased calcium and a decreased magnesium and increased calcium/magnesium ratio in hypertensive cells. In this context, in aortic smooth muscle cells from 13 spontaneously hypertensive rats (SHR) of the Münster strain (systolic blood pressure 188.4 ± 9.8 mm Hg) and 13 normotensive rats (NT, systolic blood pressure 118.5 ± 7.2 mm Hg) aged 9 months, the intracellular calcium and magnesium contents were measured under nearly in vivo conditions by electron probe microanalysis. Measurements were performed in aortic cryosections 3 μm thick; the calcium content was 124.7 ± 4.5 mmol/kg dry weight in SHR versus 110.3 ± 4.1 mmol/kg dry weight in NT (mean ± SD, P < .01 for both), the magnesium content was 35.5 ± 3.9 in SHR versus 50.1 ± 4.9 mmol/kg dry weight in NT ( P < .01 for both). The calcium/magnesium ratio was significantly increased in SHR versus NT (3.56 ± 3.9 versus 2.23 ± 0.27 [ P < .01 for both]). Thus, aortic smooth muscle cells from SHR are characterized by a markedly elevated intracellular calcium and decreased intracellular magnesium contents compared with normotensive cells. Cellular calcium and magnesium handling is disturbed in SHR aortic smooth muscle cells as it is in hypertensive blood cells. The increased calcium/magnesium ratio in hypertensive cells is a pathogenetic factor for the development of arteriosclerosis and hypertension.

Beta-catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts.

To evaluate whether cardiomyocyte membrane structure and cell/extracellular matrix adhesion alterations perturb the cadherin/catenin complex in the hypertrophic cardiomyopathy (HCM). Hypertrophic cardiomyopathic hamster (UM-X7.1 strain) and human hearts were studied by light and electron microscopy, Northern and Western blot analyses and immunohistochemistry. Intercalated disks are disorganized in both hamster and human cardiomyopathic hearts; beta-catenin is increased and accumulated in intercalated disks depriving cardiomyocyte nuclei of fundamental signals. The accumulation of beta-catenin is post-translationally regulated by an increased Wnt expression, a simultaneous decrease in glycogen synthase kinase 3beta (GSK3beta) expression and a different expression pattern of adenomatous polyposis coli (APC) isoforms. The reorganization of cell/cell adhesion in cardiomyopathic hearts is mainly contributed by the cadherin/catenin system, which is differently regulated to sustain cell structural rather than signalling needs causing considerable consequences in the determination of cardiomyocyte phenotype and clinical outcome. The accumulation of beta-catenin in intercalated disks could concur to increase myocardial wall stiffness and left ventricular end-diastolic pressure (LVEDP) in hypertrophic cardiomyopathic hamster and human hearts.

Effects of Panax notoginseng saponins on myocardial Gsα mRNA expression and ATPase activity after severe scald in rats

The aim of this study was to investigate the changes of myocardial Gsα mRNA expression and ATPase and the effects of Panax notoginseng saponins (PNS) on that after burns in rats. Wistar rats were exposed to a 95 °C water bath for 10 s to produce 30% TBSA skin-full-thickness scalds. Myocardial Gsα mRNA level, cAMP content and adenyl cyclase (AC) activities were determined with dot blotting hybridization, radioimmunoassay and indirect method, respectively. The ATPase activities in plasma membrane of cardiomyocytes and erythrocytes were measured colorimetrically. At 3, 6, 9 hours after scalds, the myocardial Gsα mRNA expression decreased significantly ( P<0.01). PNS (100, 200 mg kg −1, i.p.) markedly increased these levels ( P<0.01). The elevation was correlated significantly with PNS dose ( r=0.95, P<0.05). At 3, 6, 9, 12, 24, 48 hour after the scalds, the myocardial cAMP content and AC basal activity was reduced significantly. PNS (100, 200 mg kg −1, i.p.) increased cAMP content and enhanced AC activity markedly in comparison with the 3rd hour postburn group. The activities of (Ca 2+–Mg 2+)-ATPase and (Na +–K +)-ATPase in plasma membrane of myocardial cells and red blood cells in scald group were significantly lower than those in the normal control group ( P<0.01). PNS (100 mg kg −1, i.p.) improved these indexes significantly after scalds ( P<0.01 or 0.05). These data suggested that the effects of PNS on myocardium in burned rats involved its action to increase myocardial Gsα mRNA expression and AC activity, cAMP content as well as ATPase activities.

Naloxone for attenuation of interleukin 2 induced myocardial depression in rat hearts

To investigate the cardiac effect of interleukin-2 (IL-2) and to explore the underlying mechanism. The video tracking system and spectrofluorometric method were used to measure the cell contraction and intracellular calcium. Fura-2/AM was used as a calcium fluorescence probe. Langendorff perfusion technique was used to determine the effect of IL-2 on the intact heart. Compared with the control group, IL-2 5 U/ml, 50 U/ml significantly decreased cell contraction amplitude [(74.95+/-4.79) vs (98.09+/-5.02)%, (64.30+/-5.24) vs (97.38+/-4.05)%], peak velocity of cell shortening [(70.23+/-4.85)% vs (98.09+/-5.46)%, (61.15+/-5.20)% vs (97.38+/-6.85)%], peak velocity of cell relengthening [(71.22+/-4.79)% vs (98.32+/-6.08)%, (68.16+/-5.24)% vs (97.55+/-5.00)%] and end- diastolic cell length [(88.28+/-5.84)% vs (97.95+/-5.52)%, (84.18+/-6.52)% vs (98.94+/-6.76)%]. IL-2 (5 U/ml, 50 U/ml) also markedly inhibited intracellular calcium transient [(74.94+/-4.90)% vs (98.09+/-3.74)%,(71.00+/-5.28)% vs (97.38+/-5.52)%], and elevated end-diastolic calcium level of ventricular myocytes [(113.91+/-5.93)% vs (100.10+/-3.02)%, (119.09+/-7.12)% vs (100.52+/-6.00)%], which were attenuated by the opioid receptor antagonist naloxone (Nal,10 nmol/L). In the isolated perfused rat heart,when compared with the control group, IL-2 50 U/ml markedly decreased left ventricular developed pressure [(79.91+/-2.18) vs (93.84+/-2.94)mmHg], maximal rate of rise of left ventricular pressure [(2370.7358.29) vs (2591.50+/-62.81)mmHg] maximal rate of fall of left ventricular [-(1460.95+/-38.6) vs -(1634.24+/-54.05) mmHg/s] and heart rate [(217.35+/-10.56) vs (244.52+/-11.23) beats/min], but increased left ventricular end-diastolic pressure (11.44+/-1.02 vs 9.23+/-0.46). Pretreatment with Nal (10 nmol/L) antagonized the cardiac depression and left ventricular end-diastolic pressure elevation induced by IL-2. The cardiac effect of IL-2 is mediated by opioid receptors on the membrane of cardiomyocytes.

Hypoxic preconditioning of cardiomyocytes and cardioprotection: phophorylation of HIF-1α induced by p42/p44 mitogen-activated protein kinases is involved

Objective: To elucidate the molecular mechanisms involved in hypoxic preconditioning (HPC) of neonatal rat cardiomyocytes against hypoxia/reoxygenation (H/R) injury. Methods: Cardiomyocytes from neonatal Sprague–Dawley rats were randomly distributed into the following experimental groups: (1) HPC group: 20 min of hypoxia was performed to induce hypoxic preconditioning. Twenty four hours after HPC, cardiomyocytes were exposed to lethal hypoxia for 3 h followed by 3 h normoxia (reoxygenation). (2) Hypoxia/reoxygenation (H/R) group: cardiomyocytes were directly subjected to hypoxia (3 h) followed by reoxygenation (3 h). (3) PD98059+HPC (PD+HPC) group: cardiomyocytes were preincubated with PD98059 (a selective MEK-1/2 inhibitor, 50 μmol/l) 10 min prior to HPC. (4) BDM+HPC group: cardiomyocytes were pretreated with an activator of protein phosphatase 2,3-butanedione monoxide (BDM, 20 mmol/l) 10 min prior to HPC. (5) Control group: cardiomyocytes were incubated in cell incubator for 30 h. Viability of cardiomyocytes was assessed by MTT assay. Lactate dehydrogenase (LDH) activity in medium was determined using a LDH assay kit. Activity of p42/44 mitogen-activated protein kinases (p42/44 MAPKs) was detected using Western blotting method. SDS-PAGE mobility shift experiments were performed to determine phosphorylation of Hypoxia-inducible factor-1α (HIF-1α). Results: HPC promoted survival and membrane integrity of cardiomyocytes subjected to subsequent sustained H/R. The protective effects of HPC were completely abolished either by PD98059 [a selective inhibitor of MEK-1/2 (upstream activators of p42/44 MAPKs)], or by BDM (an activator of protein phosphatase). Western blot analysis showed activated p42/44 MAPKs in whole cell extracts from hypoxic preconditioned cardiomyocytes. SDS-PAGE mobility shift experiments showed increased phophorylation level of HIF-1α in HPC group, and the phosphorylation can be blocked by PD98059 or BDM. Conclusions: HPC protects neonatal cardiomyocytes against H/R injury by promoting cardiomyocyte survival and membrane integrity. The protective mechanism might be attributed to upregulation of HIF-1α phosphorylation which may be induced by P42/44 MAPKs.

Cellular remodeling in heart failure disrupts K(ATP) channel-dependent stress tolerance.

ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K(ATP) channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.

Cardiomyopathic features associated with muscular dystrophy are independent of dystrophin absence in cardiovasculature

The loss of dystrophin results in skeletal muscle degeneration and cardiomyopathy in patients with Duchenne muscular dystrophy. In skeletal muscle, dystrophin strengthens the myofiber membrane by linking the submembranous cytoskeleton and extracellular matrix through its direct interaction with the dystroglycan/sarcoglycan complex. In limb-girdle muscular dystrophy, the loss of the sarcoglycans in cardiovasculature leads to cardiomyopathy. It is unknown whether the absence of dystrophin in cardiomyocytes or cardiovasculature leads to the cardiomyopathy associated with primary dystrophin deficiency. We show here that the cardiomyopathic features of the utrophin/dystrophin-deficient mouse can be prevented by the presence of dystrophin in cardiomyocytes but not in cardiovasculature. Furthermore, restoration of the dystroglycans and sarcoglycans to the cardiomyocyte membrane is not sufficient to prevent cardiomyopathy. These data provide the first evidence that dystrophin plays a mechanical role in cardiomyocytes similar to its role in skeletal muscle. These results indicate that treatment of cardiomyocytes but not cardiovasculature is essential in dystrophinopathies.

Loss of intracellular dystrophin: a potential mechanism for myocardial reperfusion injury.

Because the absence of sarcolemmal dystrophin renders cardiomyocytes vulnerable to mechanical force, the present study investigated whether sarcolemmal membrane fragility upon reperfusion is associated with the loss of membrane dystrophin. Dystrophin was distributed exclusively in the sarcolemmal membrane of buffer-perfused rat cardiomyocytes, but was translocated to the myofibrils during 30 min of ischemia and then lost during reperfusion. Upon reperfusion, the membrane impermeable dye, Evans blue (EB), accumulated in cardiomyocytes depleted of dystrophin. Reperfusion with the contractile blocker 2,3-butanedione monoxime (BDM) resulted in no accumulation of EB in cardiomyocytes despite the loss of dystrophin. Upon withdrawal of BDM, however, EB accumulated in dystrophin-depleted cardiomyocytes. Loss of sarcolemmal dystrophin may be involved in the mechanism of contractile force-induced reperfusion injury.

Responding to Changes in Metabolism

Cardiac adenosine triphosphate (ATP)-sensitive potassium channels (K ATP ) respond to decreases in cellular ATP by opening, which reduces cellular excitability. Thus, K ATP channels serve as metabolic sensors. Abraham et al. calculated that the diffusion constants for the movement of ATP and adenosine diphosphate (ADP) between the cytosol and the plasma membrane of cardiomyocytes, where the K ATP channel resides, are insufficient for simple diffusion to allow the channel to respond to changes in cytosolic ATP concentrations. They show that phosphate flux through creatine kinase (CK) altered sensitivity of the K ATP channel for ATP, with ATP sensitivity highest under conditions of high flux through creatine kinase. Under hypoxic conditions, phosphoryl transfer through CK is inhibited, which leads to increased channel activity at lower concentrations of ATP than observed under normoxic conditions. In cells from mice deficient for the major cytosolic CK isoform in heart ( M-CK ), K ATP channel activity was not inhibited by application of creatine phosphate, and the animals had electrically unstable hearts in response to metabolic stress. M. R. Abraham, V. A. Selivanov, D. M. Hodgson, D. Pucar, L. V. Zingman, B. Wieringa, P. P. Dzeja, A. E. Alekseev, A. Terzic, Coupling of cell energetics with membrane metabolic sensing. J. Biol. Chem. 277 , 24427-24434 (2002). [Abstract] [Full Text]

Responding to Changes in Metabolism

Cardiac adenosine triphosphate (ATP)-sensitive potassium channels (KATP) respond to decreases in cellular ATP by opening, which reduces cellular excitability. Thus, KATP channels serve as metabolic sensors. Abraham et al. calculated that the diffusion constants for the movement of ATP and adenosine diphosphate (ADP) between the cytosol and the plasma membrane of cardiomyocytes, where the KATP channel resides, are insufficient for simple diffusion to allow the channel to respond to changes in cytosolic ATP concentrations. They show that phosphate flux through creatine kinase (CK) altered sensitivity of the KATP channel for ATP, with ATP sensitivity highest under conditions of high flux through creatine kinase. Under hypoxic conditions, phosphoryl transfer through CK is inhibited, which leads to increased channel activity at lower concentrations of ATP than observed under normoxic conditions. In cells from mice deficient for the major cytosolic CK isoform in heart (M-CK), KATP channel activity was not inhibited by application of creatine phosphate, and the animals had electrically unstable hearts in response to metabolic stress.M. R. Abraham, V. A. Selivanov, D. M. Hodgson, D. Pucar, L. V. Zingman, B. Wieringa, P. P. Dzeja, A. E. Alekseev, A. Terzic, Coupling of cell energetics with membrane metabolic sensing. J. Biol. Chem. 277, 24427-24434 (2002). [Abstract] [Full Text]

PKC Moved to the Membrane by Tyrosine Nitration

Modification of proteins by covalent attachment of NO groups has emerged as an important mechanism for the regulation of signaling. In addition, drugs that produce reactive NO species are well-known agents used during cardiac ischemic episodes. Balafanova et al. show that tyrosine nitration of protein kinase Cε (PKCε) stimulates PKCε catalytic activity by promoting its interaction with receptor for activated protein kinase C2 (RACK2), and, thus, its translocation to the membranes of cardiomyocytes. This elucidates a mechanism by which NO can stimulate PKCε activity.Z. Balafanova, R. Bolli, J. Zhang, Y. Zheng, J. M. Pass, A. Bhatnagar, X.-L. Tang, O. Wang, E. Cardwell, P. Ping, Nitric oxide (NO) induces nitration of protein kinase Cε (PKCε), facilitating PKCε translocation via enhanced PKCε-RACK2 interactions: A novel mechanism of NO-triggered activation of PKCε. J. Biol. Chem. 277, 15021-15027 (2002). [Abstract] [Full Text]

PKCε Moved to the Membrane by Tyrosine Nitration

Modification of proteins by covalent attachment of NO groups has emerged as an important mechanism for the regulation of signaling. In addition, drugs that produce reactive NO species are well-known agents used during cardiac ischemic episodes. Balafanova et al. show that tyrosine nitration of protein kinase Cε (PKCε) stimulates PKCε catalytic activity by promoting its interaction with receptor for activated protein kinase C2 (RACK2), and, thus, its translocation to the membranes of cardiomyocytes. This elucidates a mechanism by which NO can stimulate PKCε activity. Z. Balafanova, R. Bolli, J. Zhang, Y. Zheng, J. M. Pass, A. Bhatnagar, X.-L. Tang, O. Wang, E. Cardwell, P. Ping, Nitric oxide (NO) induces nitration of protein kinase Cε (PKCε), facilitating PKCε translocation via enhanced PKCε-RACK2 interactions: A novel mechanism of NO-triggered activation of PKCε. J. Biol. Chem. 277 , 15021-15027 (2002). [Abstract] [Full Text]