Plasma Membrane Of Cardiac Myocytes Research Articles

Cationic amino acid transporters and their modulation by nitric oxide in cardiac muscle cells.

Cationic amino acid transporters (CATs) play a central role in the supply of the substrate L-arginine to intracellular nitric oxide synthases (NOS), the enzymes responsible for the synthesis of nitric oxide (NO). In heart, NO produced by cardiac myocytes has diverse and even opposite effects on myocardial contractility depending on the subcellular location of its production. Approximately a decade ago, using a combination of biophysical and biochemical approaches, we discovered and characterized high- and low-affinity CATs that function simultaneously in the cardiac myocyte plasma membrane. Later on, we reported a negative feedback regulation of NO on the activity of cardiac CATs. In this way, NO was found to modulate its own biosynthesis by regulating the amount of L-arginine that becomes available as NOS substrate. We have recently solved the molecular determinants for this NO regulation on the low-affinity high-capacity CAT-2A. This review highlights some biophysical and biochemical features of L-arginine transporters and their potential relation to cardiac muscle physiology and pathology.

Dilated cardiomyopathy mutations in δ-sarcoglycan exert a dominant-negative effect on cardiac myocyte mechanical stability.

Delta-sarcoglycan is a component of the sarcoglycan subcomplex within the dystrophin-glycoprotein complex located at the plasma membrane of muscle cells. While recessive mutations in δ-sarcoglycan cause limb girdle muscular dystrophy 2F, dominant mutations in δ-sarcoglycan have been linked to inherited dilated cardiomyopathy (DCM). The purpose of this study was to investigate functional cellular defects present in adult cardiac myocytes expressing mutant δ-sarcoglycans harboring the dominant inherited DCM mutations R71T or R97Q. This study demonstrates that DCM mutant δ-sarcoglycans can be stably expressed in adult rat cardiac myocytes and traffic similarly to wild-type δ-sarcoglycan to the plasma membrane, without perturbing assembly of the dystrophin-glycoprotein complex. However, expression of DCM mutant δ-sarcoglycan in adult rat cardiac myocytes is sufficient to alter cardiac myocyte plasma membrane stability in the presence of mechanical strain. Upon cyclical cell stretching, cardiac myocytes expressing mutant δ-sarcoglycan R97Q or R71T have increased cell-impermeant dye uptake and undergo contractures at greater frequencies than myocytes expressing normal δ-sarcoglycan. Additionally, the R71T mutation creates an ectopic N-linked glycosylation site that results in aberrant glycosylation of the extracellular domain of δ-sarcoglycan. Therefore, appropriate glycosylation of δ-sarcoglycan may also be necessary for proper δ-sarcoglycan function and overall dystrophin-glycoprotein complex function. These studies demonstrate that DCM mutations in δ-sarcoglycan can exert a dominant negative effect on dystrophin-glycoprotein complex function leading to myocardial mechanical instability that may underlie the pathogenesis of δ-sarcoglycan-associated DCM.

Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure.

Action potentials (APs), via the transverse axial tubular system (TATS), synchronously trigger uniform Ca(2+) release throughout the cardiomyocyte. In heart failure (HF), TATS structural remodeling occurs, leading to asynchronous Ca(2+) release across the myocyte and contributing to contractile dysfunction. In cardiomyocytes from failing rat hearts, we previously documented the presence of TATS elements which failed to propagate AP and displayed spontaneous electrical activity; the consequence for Ca(2+) release remained, however, unsolved. Here, we develop an imaging method to simultaneously assess TATS electrical activity and local Ca(2+) release. In HF cardiomyocytes, sites where T-tubules fail to conduct AP show a slower and reduced local Ca(2+) transient compared with regions with electrically coupled elements. It is concluded that TATS electrical remodeling is a major determinant of altered kinetics, amplitude, and homogeneity of Ca(2+) release in HF. Moreover, spontaneous depolarization events occurring in failing T-tubules can trigger local Ca(2+) release, resulting in Ca(2+) sparks. The occurrence of tubule-driven depolarizations and Ca(2+) sparks may contribute to the arrhythmic burden in heart failure.

Action potential propagation in transverse-axial tubular system is impaired in heart failure

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.

Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function

The abnormal glycosylation and loss of extracellular matrix receptor function of the protein dystroglycan (DG) lead to the development of muscular dystrophy and cardiomyopathy. Dystroglycan is an important receptor for extracellular matrix proteins, such as laminin, in the basement membrane surrounding muscle. Large(myd) mice have a null mutation in a gene encoding the glycosyltransferase LARGE that results in abnormal glycosylation of α-DG and phenotypes similar to those in human α-DG glycosylation-deficient muscular dystrophy. Here, we show that Large(myd) hearts with the loss of DG extracellular matrix receptor function display a cardiomyopathy characterized by myocyte damage in patches of cells positive for membrane impermeant dyes. To examine the cellular mechanisms, we show that isolated adult cardiac myocytes from Large(myd) mice retain normal laminin-dependent cell adhesion, cell surface laminin deposition and basement membrane assembly. However, although isolated adult cardiac myocytes with the loss of α-DG glycosylation adhere normally to laminin substrates both passively and in the presence of mechanical activity, Large(myd) myocytes rapidly take up membrane impermeant dye following cyclical cell stretching. Therefore, while other cell surface laminin receptors are likely responsible for myocardial cell adhesion to the basement membrane, DG has a unique function of stabilizing the cardiac myocyte plasma membrane during repetitive mechanical activity by tightly binding the transmembrane dystrophin-glycoprotein complex to the extracellular matrix. This function of DG to stabilize the myocyte membrane during normal physiologic cell length changes is likely critical for the prevention of the myocardial damage and subsequent remodeling observed in α-DG glycosylation-deficient muscular dystrophies.

Adenosine A1 receptors induce translocation of PKCε to the caveolar membrane of cardiac myocytes

Caveolae are specialized microdomains of the plasma membrane. They play an important role in the cardioprotective effect of ischemic preconditioning. Both adenosine receptors and PKCε, which have been shown to reside in the caveolae of cardiac myocytes, are associated with ischemic preconditioning. However, the potential link between adenosine receptors and PKCε has not been elucidated. The present study was designed to test the hypothesis that adenosine A1 receptors induce the translocation of PKCε to the plasma membrane within caveolae. Immunoblot analysis from the homogenate of freshly isolated cardiac myocytes revealed that the intensity of 100 kD band corresponding to PKCε increased significantly in the membrane fractions in cells pretreated with adenosine (100 μM) when compared with non‐stimulated cells. The effect of adenosine on PKCε translocation was blocked by a specific A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 1 μM). Western blot in caveolin‐enriched fractions prepared from isolated cardiac myocytes showed that immunoreactivity for PKCε increased significantly from cells treated with adenosine but not with adenosine plus DPCPX. Furthermore, PKCε was detected in the anti‐caveolin‐3 immunoprecipitates from caveolar fractions. In conclusion, we demonstrate that activation of adenosine A1 receptors promotes the translocation of PKCε to the caveolin‐enriched plasma membrane of cardiac myocytes.

Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes

ATP-sensitive potassium (K(ATP)) channels in the heart are critical regulators of cellular excitability and action potentials during ischaemia. However, little is known about subcellular localization of these channels and their regulation. The present study was designed to explore the potential role of caveolae in the regulation of K(ATP) channels in cardiac ventricular myocytes. Both adult and neonatal rat cardiomyocytes were used. Subcellular fractionation by density gradient centrifugation, western blotting, co-immunoprecipitation, and immunofluorescence confocal microscopy were employed in combination with whole-cell voltage clamp recordings and siRNA gene silencing. We detected that the majority of K(ATP) channels on the plasma membrane of cardiac myocytes were localized in caveolin-3-enriched microdomains by cell fractionation and ultracentrifugation followed by western blotting. Immunofluorescence confocal microscopy revealed extensive colocalization of K(ATP) channel pore-forming subunit Kir6.2 and caveolin-3 along the plasma membrane. Co-immunoprecipitation of cardiac myocytes showed significant association of Kir6.2, adenosine A(1) receptors, and caveolin-3. Furthermore, whole-cell voltage clamp studies suggested that adenosine A(1) receptor-mediated activation of K(ATP) channels was largely eliminated by disrupting caveolae with methyl-beta-cyclodextrin or by small interfering RNA, whereas pinacidil-induced K(ATP) activation was not altered. We demonstrate that K(ATP) channels are localized to caveolin-enriched microdomains. This microdomain association is essential for adenosine receptor-mediated regulation of K(ATP) channels in cardiac myocytes.

The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes

Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.

VDAC‐1 May Interact with CFTR to Impart Important Cellular Function in Mouse Heart

The cystic fibrosis transmembrane-conductance regulator (CFTR) Cl− channels may play an important role in ischemic preconditioning (IPC)-mediated cardioprotection in mouse heart. To investigate the mechanisms for the role of CFTR in IPC we used proteomic approaches to analyze changes in the protein expression profiles of both plasma membrane (PM) and mitochondrial (MT) compartments caused by CFTR gene knockout. Proteins from heart tissue (10 hearts pooled as one sample) of either wild-type (FVB/NJ) or CFTR−/− (FABPCFTR) mice were isolated, purified, and fractionated. Marker enzyme assays demonstrated a 1.74-fold enrichment of PM in the PM fractions and a 4.4-fold enrichment of MT in MT fractions, and a strong reduction of other compartments. Proteomic analysis (2D-PAGE, MALDI-TOF/TOF mass spectrometry) unambiguously identified a significant (40%) decrease in the expression of the voltage-dependent anion channel 1 (VDAC-1) in cardiac PM proteins of the CFTR−/− mice. Immunocytochemistry confirmed VDAC-1 expression in PM of cardiac myocytes. Western blots on both 2D-PAGEs and 1D gels further confirmed the reduced expression of VDAC-1 in PM of CFTR−/− mouse heart. These results strongly suggest that VDAC-1 may be an important component of the CFTR subproteome and may interact with CFTR to impart orchestrated functions in IPC-mediated cardioprotection. (Supported by NCRR at NIH P20 RR15581 and NIH R01 HL63914)

Combined biochemistry and histocytochemistry as a tool to investigate Ecto-ATPase in the cardiac muscle.

Ecto-ATPase (ecto-adenosine triphosphatase), a key enzyme of cardiac metabolism, is responsible for modulation of the concentration of extracellular nucleotides in the heart. We present methodology consisting of the combined use of biochemical and histocytochemical techniques to study its properties. Using samples from essentially the same preparation, we applied biochemistry and histocytochemistry to determine biochemical characteristics of ecto-ATPase and an in situ localization of its reactivity. Our results indicated that detected enzyme resists fixation, depends on divalent ions, and hydrolyzes ATP, but not AMP or ADP-beta-S. Reaction product of the enzyme activity was found confined to the extracellular surface of the plasma membrane of cardiac myocytes and endothelial cells due to the corresponding orientation of the enzyme active sites. Experiments using an inhibitor justified specificity of the reaction. When used together with molecular biological and immunocytochemical techniques, the present methodological approach should be capable of yielding important information about the actual ability of ecto-ATPase to operate.

Evaluation of the Safety and Cardioprotective Effects of Eniporide, a Specific Sodium/Hydrogen Exchange Inhibitor, Given as Adjunctive Therapy to Reperfusion in Patients with Acute Myocardial Infarction

Background: From the results of various experimental studies, increasing evidence has been accumulated that the activity of the Na⁺/H⁺ exchanger (NHE) plays an important role in the unfavorable sequels of myocardial ischemia and reperfusion. Eniporide specifically inhibits the NHE-1 isoform on the cardiac myocyte plasma membrane and has been shown to reduce myocardial cell death and limit infarct size in experimental infarct models. Objective: The objective of the ESCAMI trial is to investigate the tolerability and cardioprotective efficacy of eniporide as an adjunct to reperfusion therapy in patients with acute myocardial infarction (AMI) compared to reperfusion therapy alone. Methods: In this phase II dose-finding trial, patients with AMI ≤6 h in duration and ST elevation in at least two leads are randomized to receive either eniporide or placebo as an adjunctive therapy to thrombolysis or primary percutaneous transluminal coronary angioplasty (PTCA). Eniporide or placebo has to be administered as a 10-min infusion before the initiation of the reperfusion therapy. The primary efficacy end point is the cumulative release of α-hydroxybutyrate dehydrogenase (area under the curve from 0 to 72 h). A total of 10 blood samples are taken over 72 h (baseline, 4, 8, 12, 16, 24, 36, 48, 60 and 72 h) and analyzed by a central laboratory. In addition, other cardiac markers, including creatine kinase (CK), CK-MB and troponin T and I are examined. Secondary target variables include relevant cardiovascular events within 6 weeks and total mortality up to 6 months. Resolution of ST segment elevation is measured at 90 and 180 min after the start of thrombolysis and immediately and 90 min after PTCA. The combined clinical end point at 6 weeks consists of total mortality, the occurrence of heart failure, cardiogenic shock or sustained ventricular arrhythmia. For the safety evaluation of the new drug, the incidence and type of adverse events is recorded up to 6 weeks. A two-stage adaptive design is employed that allows a reduction in the number of patients treated with ineffective doses compared to conventional dose-finding studies. In stage 1, four doses of eniporide (50, 100, 150 and 200 mg) are compared to placebo. The objective of stage 1 is to obtain some initial evidence of efficacy and to select a subset of doses to be tested in stage 2. A linear trend test is employed to test the efficacy at stage 1. In stage 2, the efficacy of the best two doses selected from stage 1 are compared to placebo. Conclusion: The ESCAMI trial tests a novel pharmacologic agent (eniporide) in patients with AMI and the hypothesis that inhibition of the NHE system before reperfusion can decrease infarct size and improve the clinical outcome. In addition, this trial is designed as a phase II trial and a two-stage design is employed to decrease the number of patients treated with an ineffective dose. This trial will add to our knowledge about the concept of myocardial cell protection by NHE inhibition induced before reperfusion therapy and the potential of this treatment strategy for clinical practice.

Sodium ion/hydrogen ion exchange inhibition: a new pharmacologic approach to myocardial ischemia and reperfusion injury.

Over the past few years, it has been shown that the cardiac myocyte plasma membrane sodium ion/hydrogen ion exchanger (NHE) plays an important role in the maintenance of intracellular pH, sodium, and calcium ion homeostasis. From the results of various experimental studies, it is clear that this ion exchanger is an important mediator of ischemic-reperfusion injury of the heart. During myocardial ischemia, intracellular acidosis develops quickly, activating the exchanger to extrude H+ into the extracellular environment and bring Na+ into the cell. With further progression of ischemia, the cell is unable to handle the overload of Na+, causing it to use its Na+/Ca2 exchanger to unload intracellular Na+ into the extracellular space. At the same time, however, calcium is being transported into the cell. This can lead to detrimental cardiac injury, such as contracture and necrosis. During myocardial reperfusion, these events are magnified because the return of blood flow lowers the extracellular H+ concentration, stimulating the NHE to extrude more intracellular H+ ion. This leads to intracellular Na+ excess and eventually, intracellular Ca2+ overload and cardiac injury. In an effort to alter these pathophysiologic events, a number of investigators have studied the ability of various NHE inhibitors, such as amiloride, analogues of amiloride, and other drugs (HOE 694, HOE 642), to prevent cardiac ischemic-reperfusion damage. Preliminary results from studies in animal models have revealed that most of these agents are able to attenuate the development of myocardial contracture, infarction, and arrhythmias during both ischemia and reperfusion. Their efficacy and cardioprotective effects in human beings have yet to be determined. These agents appear to be promising not only in the prevention and treatment of ischemic heart disease, but also in avoiding cardiac damage in situations where low-flow states are followed by immediate recovery of flow, as in coronary artery bypass graft surgery, percutaneous transluminal coronary angioplasty, thrombolytic therapy, and coronary arterial vasospasm. This article reviews the physiology of the NHE and analyzes the potential role of NHE inhibitors in the prevention of ischemic-reperfusion injury and other cardiac disease states.

Contraction-Induced Cell Wounding and Release of Fibroblast Growth Factor in Heart

The heart hypertrophies in response to certain forms of increased mechanical load, but it is not understood how, at the molecular level, the mechanical stimulus of increased load is transduced into a cell growth response. One possibility is that mechanical stress provokes the release of myocyte-derived autocrine growth factors. Two such candidate growth factors, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively), are released via mechanically induced disruptions of the cell plasma membrane. In the present study, we demonstrate that transient, survivable disruption (wounding) of the cardiac myocyte plasma membrane is a constitutive event in vivo. Frozen sections of normal rat heart were immunostained to reveal the distribution of the wound event marker, serum albumin. Quantitative image analysis of these sections indicated that an average of 25% of the myocytes contained cytosolic serum albumin; ie, this proportion had suffered a plasma membrane wound. Wounding frequency increased approximately threefold after beta-adrenergic stimulation of heart rate and force of contraction. Heparin-Sepharose chromatography, enzyme-linked immunosorbent assay, growth assay coupled with antibody neutralization, and two-dimensional SDS-PAGE followed by immunoblotting were used to demonstrate that both aFGF and bFGF were released from an ex vivo beating rat heart. Importantly, beta-adrenergic stimulation of heart rate and force of contraction increased FGF release. Cell wounding is a fundamental but previously unrecognized aspect of the biology of the cardiac myocyte. We propose that contraction-induced cardiac myocyte wounding releases aFGF and bFGF, which then may act as autocrine growth-promoting stimuli.

Action of metformin on glucose transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy and diabetic rats.

The effects of the antidiabetic drug metformin on glucose transport were investigated in freshly isolated heart muscle cells from healthy and streptozotocin-diabetic rats. In vivo treatment of diabetic rats with metformin failed to affect the basal and insulin-stimulated rate of glucose transport measured in isolated cells. In vitro exposure to therapeutic concentrations (< or = 10(-4) M) of metformin did not influence glucose transport, even upon incubation times up to 5 h or in the presence of high glucose (20 nM). In contrast, higher metformin concentrations produced an 8- to 12-fold increase in glucose uptake (with a lag of 90 min, and a maximum at 180 min and approximately 5 mM). In the presence of submaximal insulin concentrations (< or = 3.10(-10) M), the effects of metformin (5 mM) and of insulin were more than additive, whereas, at saturating insulin concentrations (10(-8) M), partial additivity was observed. Like insulin, metformin caused an approximately 1.6-fold increase in the content of both glucose transporter isoforms GLUT1 and GLUT4 in the plasma membrane of cardiac myocytes, with a corresponding decrease in an intracellular membrane fraction. cAMP-elevating treatments depressed the metformin-, but not the insulin-dependent glucose uptake, by 20-30%. In myocytes from diabetic rats, the rate of metformin-activated glucose transport was similar to that of cells from control animals, whereas basal and insulin-stimulated transport were substantially diminished. Finally, metformin (5 mM) induced a slight depression of oxygen consumption and energy metabolism of myocytes (as determined by measuring their level of energy-rich phosphates) comparable to the effects of hypoxia in rat hearts. In conclusion, these data do not provide evidence in favor of the hypothesis that glucose uptake by muscle tissue represents the site of metformin's therapeutic action in vivo. On the other hand, the large, insulin-independent effect of metformin at high concentrations (approximately mM) in vitro may be related to the action of hypoxia and occurs through a redistribution of glucose carriers from an intracellular locus to the plasma membrane. The mechanism (or signal) involved in metformin's action is likely to differ from that triggered by insulin and is not impaired in the diabetic state.

Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes.

Acute myocardial ischemia is accompanied by an increase in glucose uptake and metabolism, which appears to be important in protecting myocardial cells from irreversible ischemic injury. Because insulin augments myocardial glucose uptake by inducing the translocation of glucose transporters from an intracellular compartment to the plasma membrane, we hypothesized that acute ischemia would trigger a similar translocation. We used a subcellular fractionation method to separate intracellular membrane and plasma membranes from control, ischemic, and hypoxic Langendorff-isolated perfused rat hearts and determined the expression of the major myocardial glucose transporter, GLUT4, in these separated membrane fractions. We found that translocation of GLUT4 molecules occurred in ischemic, hypoxic, and insulin-treated hearts and in hearts that underwent ischemia plus insulin treatment. The percentages of GLUT4 molecules present on the plasma membrane in the different conditions were as follows: control, 18.0 +/- 2.8%; ischemia, 41.3 +/- 9.4%; hypoxia, 31.1 +/- 2.9%; insulin, 61.1 +/- 2.6%; and ischemia plus insulin, 66.8 +/- 5.7%. Among the statistically significant differences in these values were the difference between control and ischemia and the difference between ischemia alone and insulin plus ischemia. Ischemia causes substantial translocation of GLUT4 molecules to the plasma membrane of cardiac myocytes. A combination of insulin plus ischemia stimulates an even greater degree of GLUT4 translocation. GLUT4 translocation is likely to mediate at least part of the increased glucose uptake of ischemic myocardium and may be a mechanism for the cardioprotective effect of insulin during acute myocardial ischemia.

Rat atrial myocyte plasmalemmal caveolae in situ. Reversible experimental increases in caveolar size and in surface density of caveolar necks.

The structure, size, and surface density of the conspicuous flask-shaped structures called caveolae that are located under the plasma membrane of cardiac myocytes in intact rat atria were studied by electron microscopy after physiological perturbations designed to examine whether caveolae and/or their necks are fixed or mobile and whether the caveolar lumen is always open or can close off from the interstitial space. We showed that, in stretched and unstretched atria, horseradish peroxidase could enter or be washed out of caveolae at 37 degrees, 18 degrees, and 4 degrees C, but this finding does not rule out that caveolae and/or their necks can cycle rapidly between states closed and open to the interstitial space. Electron microscopy of thin sections revealed that exposure of atria at 37 degrees or 18 degrees C to physiological salt solutions made hypertonic by adding 150 mM sucrose or mannitol resulted in a striking enlargement of caveolar profiles within 1 to 5 minutes. Caveolar enlargement was rapidly reversible on return to control saline. After freeze fracture of atria exposed to these hypertonic solutions, quantitative analysis of electron micrographs of the fracture faces revealed statistically significant increases in cross-sectional diameter of cross-fractured caveolar necks and in mean number of caveolar necks penetrating per unit area of plasmalemmal fracture face. These results suggest that atrial myocyte caveolae are dynamic structures whose necks may be reversibly inserted into and withdrawn from the plasmalemma, possibly (but not necessarily) corresponding to states in which caveolae are, respectively, open and closed to the interstitial spaces.

The Ca-channel as cardiac autoantigen.

We recently described autoantibodies to the ADP/ATP carrier of the inner mitochondrial membrane as an organ specific autoantigen found in high frequencies in sera of patients with myocarditis and dilated cardiomyopathy. These antibodies not only showed an intracellular binding at the mitochondrial membrane, but also crossreact with antigenic determinants at the cardiac myocyte plasma membrane. They therefore may account, at least in part, for the so-called sarcolemmal-bound IgG of heart-reactive autoantibodies found to be associated with these diseases. Heart muscle diseases are associated with electrocardiographic abnormalities such as complex ventricular arrhythmias and disturbed ventricular function. Our data indicate that abnormalities in current spread and impaired heart contractility might also be caused by an antibody mediated disturbance of calcium channel gating leading to prolongation of the action potential and potentiation of tension. The enhanced calcium permeability of the cell may moreover cause calcium overload and cell death. The calcium channel complex plays a central role in cardiac contractility. Disturbing calcium channel gating by antibodies might therefore contribute to the pathogenesis of these diseases.

Changes of plasma membrane of cardiac myocytes produced by treatment with hyaluronidase

Changes of plasma membrane of cardiac myocytes produced by treatment with hyaluronidase