Selective elimination of I(K,slow1) in mouse ventricular myocytes expressing a dominant negative Kv1.5alpha subunit

Although previous studies have revealed a role for the voltage-gated K+ channel alpha-subunit Kv1.5 (KCNA5) in the generation of the 4-aminopyridine (4-AP)-sensitive component of delayed rectification in mouse ventricles (IK,slow1), the phenotypic consequences of manipulating IK,slow1 expression in vivo in different (mouse) models are distinct. In these experiments, point mutations were introduced in the pore region of Kv1.5 to change the tryptophan (W) at position 461 to phenylalanine (F) to produce a nonconducting subunit, Kv1.5W461F, that is shown to function as a Kv1 subfamily-specific dominant negative (Kv1.5DN). With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, three lines of Kv1.5DN-expressing (C57BL6) transgenic mice were generated and characterized. Electrophysiological recordings from Kv1.5-DN-expressing left ventricular myocytes revealed that the micromolar 4-AP sensitive IK,slow1 is selectively eliminated. The attenuation of IK,slow1 is accompanied by increased ventricular action potential durations and marked QT prolongation. In contrast to previous findings in mice expressing a truncated (DN) Kv1.1 transgene; however, no electrical remodeling is evident in Kv1.5DN-expressing ventricular myocytes, and the (Kv1.5DN-induced) elimination of IK,slow1 does not result in spontaneous ventricular arrhythmias.     

Voltage-dependence of contraction in streptozotocin-induced diabetic myocytes

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of –40 mV, test pulses were applied at potentials between –30 and +50 mV in 10 mV increments. L-type Ca²⁺ current (I _Ca,L) density and contraction were measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The I _Ca,L density was significantly (p < 0.05) reduced throughout voltage ranges (–10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between –20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca²⁺ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca²⁺ trigger through L-type-Ca²⁺-channels and lead to normalised contraction. (Mol Cell Biochem 261: 235–243, 2004)     

Voltage-dependence of contraction in streptozotocin-induced diabetic myocytes

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of -40 mV, test pulses were applied at potentials between -30 and +50 mV in 10 mV increments. L-type Ca2+ current (I Ca,L) density and contraction were measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The I Ca,L density was significantly (p < 0.05) reduced throughout voltage ranges (-10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between -20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca2+ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca2+ trigger through L-type-Ca2+-channels and lead to normalised contraction.     

Selective Oma1 protease-mediated proteolysis of Cox1 subunit of cytochrome oxidase in assembly mutants

Stalled biogenesis of the mitochondrial cytochrome c oxidase (CcO) complex results in degradation of subunits containing redox cofactors. The conserved Oma1 metalloproteinase mediates facile Cox1 degradation in cells lacking the Coa2 assembly factor, but not in a series of other mutants stalled in CcO maturation. Oma1 is activated in coa2Δ cells, but the selective Cox1 degradation does not arise merely from its activation. Oma1 is also active in cells with dysfunctional mitochondria and cox11Δ cells impaired in CcO maturation, but this activation does not result in Oma1-mediated Cox1 degradation. The facile and selective degradation of Cox1 in coa2Δ cells, relative to other CcO assembly mutants, is likely due to impaired hemylation and subsequent misfolding of the subunit. Specific Cox1 proteolysis in coa2Δ cells arises from a combination of Oma1 activation and a susceptible conformation of Cox1.     

Selective modification of an alpha subunit of chloroplast coupling factor 1

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

MVB-12, a fourth subunit of metazoan ESCRT-I, functions in receptor downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis.     

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and β3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIla), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIla bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3–15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12–25-fold more than Fn. FXIIla bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10–20-fold less than Fn. The K _d s for FXIIla binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The K _d for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn]>[FXIIIa-Fgn]=[FXIIIa-Fbn]>[FXIII-Fbn]>[FXIIIFgn]=[FXIIIa-Fn]>[Tg-Fbn]=[Tg-Fgn]>[FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.Abbreviations Tg cellular transglutaminase - FXIII coagulation factor XIII - FXIIla factor XIIIa (thrombin-activated FXIII) - Fgn human plasma fibrinogen - Fn human plasma fibronectin - Fbn human plasma fibrin (thrombin-cleaved fibrinogen) - ECM extracellular matrix     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

Hypoxia-induced shortening of the action potential duration, attributed to activation of the ATP-sensitive potassium (K_ATP) channels, occurs to a much greater extent in ventricular cells from diabetic rats. This study examined whether the K_ATP channels are altered in streptozotocin-diabetic myocardium. In inside-out patches from ventricular myocytes (with symmetrical 140 mM [K+]), inward K_ATP currents (at potentials negative to the K+ reversal potential) were similar in amplitude in control and diabetic patches (slope conductances: 69 and 74 pS, respectively). However, outward single-channel currents were larger for channels from diabetic heart cells than from control cells (e.g., at +75 mV the diabetic channel currents were 3.7 ± 0.3 pA vs. 2.7 ± 0.1 pA for control currents, p < 0.05), due to reduced inward rectification of diabetic channel currents. There was no difference in open and closed times between control and diabetic channels. The IC₅₀ for ATP inhibition of the K_ATP channel single-channel currents was 11.4 M for control currents and 4.7 M for diabetic channel currents. Thus, the major difference found between K_ATP channels from control and diabetic hearts was the greater outward diabetic single-channel current, which may contribute to the enhanced sensitivity to hypoxia (or ischemia) in diabetic hearts.     

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.     

Antisense oligonucleotide against the Ca channel beta subunit decreases L-type Ca current in rat ventricular myocytes

The role of endogenous beta subunits of the L-type Ca channel in native cardiac ventricular myocytes is unclear. We have therefore investigated the effect of inhibiting beta subunit expression in rat myocytes, by culturing isolated myocytes for 24 h with either antisense oligonucleotide against the beta subunit or with scrambled oligonucleotide (control). Alpha1 subunit expression and distribution were then determined by immunolabeling, and L-type Ca current measured using the whole cell patch-clamp technique. Cells treated with antisense showed increased perinuclear staining for alpha1, decreased Ca current amplitude and a small rightward shift of the activation curve and the I-V relation, with no significant effect on inactivation. These data suggest that endogenous beta subunits in native cardiac myocytes help to traffic the alpha1 subunit to the cell membrane and thus play a major role in determining Ca current amplitude.