Alterations of sodium channel kinetics and gene expression in the postinfarction remodeled myocardium.

INTRODUCTION: After a myocardial infarction (MI), the heart undergoes a remodeling process that includes hypertrophy of noninfarcted left ventricular myocytes. Alterations in the genetic expression, including reexpression of fetal isogene patterns, can result in electrophysiologic changes that contribute to the arrhythmogenicity of post-MI heart. The present study investigated possible alterations in gene expression of Na+ channel subtypes, as well as the kinetics of the Na+ current (I(Na)), in 3- to 4-week-old post-MI rat remodeled left ventricular myocardium. METHODS AND RESULTS: Using a macropatch technique, we showed increased Na+ channel bursting activity during sustained depolarization in post-MI remodeled myocytes resulting in a large slow component of the I(Na) decay. A tetrodotoxin-sensitive current contributed 18% to the prolonged APD90 of isolated post-MI myocytes compared with 6% in control myocytes. Our molecular studies revealed that, in addition to the rat heart I (rH I) subtype, thought to be the predominant subtype that encodes a tetrodotoxin-resistant isoform, the brain subtypes NaCh I and NaCh Ia also are expressed in the rat myocytes. Post-MI remodeled myocardium showed increased expression of NaCh I protein with reversion of the NaCh Ia/NaCh I isoform ratio toward the fetal phenotype. CONCLUSION: Our findings raise the possibility that the increase in the slow component of I(Na) in post-MI remodeled myocytes is secondary to the increased expression of NaCh I. Additional studies are required to address these questions and to characterize the functional role of the NaCh I subtypes in cardiac myocytes.

Alterations in cardiac gene expression during ventricular remodeling following experimental myocardial infarction.

Following myocardial Infarction (MI) the heart undergoes a process of remodeling characterized by considerable hypertrophy of the non-infarcted myocardium. We have recently characterized the molecular basis of key electrophysiologic alterations that may provide insight into the arrhythmogenecity of post-MI remodeled hypertrophied myocardium. To further characterize other key alterations in the pattern of cardiac gene expression in a time-dependent manner, we have measured mRNA and immunoreactive protein levels of selective cardiac genes in the remodeled hypertrophied left-ventricular (LV) myocardium of rats, 3 and 21 days after left-coronary ligation and compared the results with sham-operated rats. RNase protection assay was performed to assess the expression of c-fos, atrial natriuretic factor (ANF), brain natriuretic factor (BNF), alpha2/3 isoform of Na-K ATPase, cardiac alpha/beta isoform of myosin heavy chain (MHC). Compared to the sham group, the expression of c-fos was increased 10-fold (P<0.02) in the MI group on day 3, but unlike other overload hypertrophy models, the expression remained elevated by three-fold on day 21. Similar to other overload models, the ANF and BNF expression increased significantly. No alterations were observed in the expression of cardiac alpha-actin. There was reexpression of the fetal isogene form of MHC and Na-K ATPase after MI. The beta-MHC mRNA levels, the fetal isoform of MHC, returned to basal levels after 21 days. After an initial five-fold decrease the adult isoform of alphaNa-K ATPase, alpha2 Na-K ATPase mRNA, returned to control levels and similar changes were seen in the corresponding protein levels. These findings indicate that during LV remodeling and hypertrophy following MI, there is an upregulation of early response genes and fetal isogene expression. The pattern of activation, however, is distinct from that observed in other overload models, indicating the possible involvement of alternate signal transduction pathways.

Differential expression of voltage-gated K+ channel genes in left ventricular remodeled myocardium after experimental myocardial infarction.

Left ventricular (LV) remodeling after experimental myocardial infarction (MI) is associated with hypertrophy of noninfarcted myocardium and electrophysiological alterations. We have recently shown that post-MI hypertrophied LV myocytes have prolonged action potential duration (APD) and generate triggered activity from early afterdepolarizations. The prolonged APD was attributed to decreased density of the two outward K+ currents, I(to)-fast (I(to)-f) and I(to)-slow (I(to)-s), rather than changes in the density and/or kinetics of the L-type Ca2+ current. The changes in ionic current density may be related to alterations in the expression and levels of ion channel proteins. To test this hypothesis, rats underwent either left anterior descending coronary artery (LAD) ligation (post-MI group [n = 10]) or sham surgery (sham group [n = 10]). Three weeks later transcripts from the noninfarcted LV myocardium in the post-MI group (n = 6) and LV myocardium of the sham group (n = 6) were analyzed by RNase protection assay. Expressions of five K+ channel subunit mRNAs (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) reported in the rat ventricle were analyzed. Compared with the sham group, expressions of Kv1.4, Kv2.1 (putative I(to)-s), and Kv4.2 (putative I(to)-f) channel subunit mRNAs were significantly decreased by 60% (P < .03), 54% (P < .005), and 53% (P < .002), respectively, in the post-MI group. There was no significant change in the Kv1.2 and Kv1.5 mRNA levels. Western blotting demonstrated a similar decrease in the Kv2.1 and Kv4.2 immunoreactive protein levels (43% [P < .03] and 67% [P < .003], respectively [n = 4]) and no significant change in Kv1.5 immunoreactive protein level. Our results strongly correlate with the electrophysiological findings in this model and show that transcriptional regulation in the post-MI remodeled rat LV is distinct for each voltage-gated K+ channel subunit. These findings provide, at least in part, the molecular basis for the electrophysiological alterations observed in this model.

Reemergence of the fetal pattern of L-type calcium channel gene expression in non infarcted myocardium during left ventricular remodeling.

The cardiac L-type voltage-dependent calcium channel (VDCC) is a critical component of cardiac action potential and excitation-contraction coupling. The objective of the present study was to examine the changes in expression in Motif IV, an alternatively spliced region of the alpha-1 subunit of the VDCC channel in postmyocardial infarction (MI) remodeled rat left ventricle. RNase protection assay was used to determine alteration in isoform expression in the noninfarcted hypertrophied ventricular myocardium 21 days post myocardial infarction. Our study demonstrates that cardiac hypertrophy is associated with significant increase in the mRNA level of the fetal isoform, with the reversion of fetal:adult isoform ratio to the fetal phenotype. Changes in isoform expression in the post-MI remodeled ventricle, not previously reported, is a pertinent genetic marker of cardiac hypertrophy.

Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements.

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants.

Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver. Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood. Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala). The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase. By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme. There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity. The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax. These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding.

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli. Role of ASP-118 and ASP-121 in catalysis.

A cDNA encoding human liver fructose-1,6-bisphosphatase was isolated from a lambda gt11 library by screening with a rat liver fructose-1,6-bisphosphatase cDNA. The cDNA (1421 base pairs) contains an open reading frame encoding 337 amino acids, corresponding to a protein with an estimated molecular weight of 36,697. Its primary sequence is highly homologous to that of the pig kidney and rat liver enzymes. The human liver cDNA was used to construct a T7 RNA polymerase-transcribed expression vector, and the enzyme was expressed in Escherichia coli BL21 (DE3). Approximately 50% of the expressed human fructose-1,6-bisphosphatase was soluble and enzymatically active, and the enzyme was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and substrate/AMP elution from carboxymethyl-Sephadex. Expressed human liver fructose-1,6-bisphosphatase had a specific activity (9.8 mumol/min/mg of protein) that was half that of the rat liver enzyme, but had an identical Km for substrate. However, the human enzyme was more sensitive to inhibition by fructose-2,6-bisphosphate (Ki = 0.3 microM) and AMP (Ki = 12 microM) than the rat liver form (fructose 2,6-P2, Ki = 4 microM; AMP, Ki = 40 microM). Crystallographic analyses have suggested that Asp-118 and Asp-121 are catalytic residues located in a negatively charged pocket that binds divalent metal cations. These residues were mutated to alanine, and the E. coli-expressed mutant enzymes were purified to homogeneity. The Asp-118-->Ala and Asp-121-->Ala mutants had 1/5000 and 1/20,000 lower Kcat values than the wild-type enzyme, respectively, consistent with their critical role in fructose-1,6-bisphosphatase catalysis.

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.