Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca2+-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca2+-concentration (increased Ca2+-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type β-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca2+-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca2+-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca2+-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, α-tropomyosin and myosin binding protein-C are considered in detai

Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constants K1 = ∼5 × 106 and K2 = ∼1 × 105 M−1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine induced cell death

Pancreatic β-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8, is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of wild-type and global ZnT8-/- mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, wild-type islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNFα, IFNγ, IL1β) treatment induced islet cell death, but to a lesser extent in islets from ZnT8-/- mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in wild-type islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8-/- mice as compared to wild-type islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8-/- mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man

Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca2+-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca2+-concentration (increased Ca2+-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type β-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca2+-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca2+-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca2+-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, α-tropomyosin and myosin binding protein-C are considered in detai

Cloning of the rat utrophin and characterization of an alternate transcript

Utrophin is an autosomal homologue of dystrophin. The dystrophin gene is located on the X-chromosome, and its product, dystrophin, is missing in muscle fibres of patients with Duchenne muscular dystrophy. The absence of dystrophin is probably the cause for this dystrophy. The gene for the human utrophin protein was cloned recently and the deduced amino acid sequence shows a significant similarity to dystrophin (Tinsley et al., Nature, 360, 591- 3, 1992). It has also a similar structure with four domains, the actin binding domain, the spectrin-like rod domain, the cysteine rich (CR) domain and the C-terminal (CT) domain. The actin binding and the CR-CT domains exhibit 85% amino acid sequence similarity between dystrophin and utrophin. With 60% similarity the rod domain is less conserved. A rat cDNA library was screened with probes originating from mouse utrophin cDNAs. Several positive clones were found and characterized. Three clones were found to overlap and to contain the whole coding region for the utrophin protein. The combined cDNA contains an open reading frame of 10 254 bp and encodes a protein of 3419 amino acids. The rat utrophin exhibits a high amino acid sequence similarity of 91% over the entire protein towards the human utrophin. Two further clones were characterized. They contain a beginning (1.8 kb) identical to the 5’ rat utrophin sequence while their ends (0.3 and 0.1 kb) are identical among themselves but different from the rat utrophin. Preliminary Northern blots and results from PCR reactions support the notion that these two clones represent a short alternative transcript of the rat utrophin Recently the finding of a small (62 kDa) protein was reported, which reacts with two monoclonal antibodies. The antibodies are directed against the Nterminus of the human utrophin, but they also recognize the rat/ mouse utrophin (Nguyen et al., FEBS Letts, 358, 262-6, 1995). The size of the protein and the tissue distribution would correlate with the results from the Northern blots

Sulfonylurea induced beta-cell apoptosis in cultured human islets

Loss of beta-cell mass and function raises a concern regarding the application of sulfonylureas for the treatment of type 2 diabetes because previous studies have shown that agents that cause closure of inwardly rectifying K(+) sulfonylurea receptor subtype of ATP-sensitive potassium channels, such as tolbutamide and glibenclamide, induce apoptosis in beta-cell lines and rodent islets. Therefore, we investigated the effect of the new insulin secretagogues, repaglinide and nateglinide, and the sulfonylurea, glibenclamide, on beta-cell apoptosis in human islets. Human islets from six organ donors were cultured onto extracellular matrix-coated plates and exposed to glibenclamide, repaglinide, or nateglinide. The doses of the three compounds were chosen according to detected maximal effects, i.e. efficacy. Exposure of human islets for 4 h to 0.1 and 10 microm glibenclamide induced a 2.09- and 2.46-fold increase in beta-cell apoptosis, respectively, whereas repaglinide (0.01 and 1 microm) did not change the number of apoptotic beta-cells. At low concentration (10 microm), nateglinide did not induce beta-cell apoptosis. However, at high concentration of 1000 microm, it induced a 1.49-fold increase in the number of apoptotic beta-cells. Prolonged exposure for 4 d of the islets to the secretagogues induced beta-cell apoptosis. The increase was of 3.71- and 4.4-fold at 0.1 and 10 microm glibenclamide, 2.37- and 3.8-fold at 0.01 and 1 microm repaglinide, and of 3.2- and 4.6-fold at 10 and 1000 microm nateglinide, respectively. Glibenclamide at 0.1-10 nm (doses that were less efficient on insulin secretion) did not induce beta-cell apoptosis after 4 h incubation as well as 0.1 nm after 4 d incubation. However, 1 and 10 nm glibenclamide for 4 d induced a 2.24- and 2.53-fold increase in beta-cell apoptosis, respectively. Taken together, closure of the inwardly rectifying K(+) sulfonylurea receptor subtype of ATP-sensitive potassium channels induces beta-cell apoptosis in human islets and may precipitate the decrease in beta-cell mass observed in patients with type 2 diabetes

Increasing complexity of the dystrophin-associated protein complex

Duchenne muscular dystrophy is a severe X chromosome-linked, muscle-wasting disease caused by lack of the protein dystrophin. The exact function of dystrophin rem to be determined. However, analysis of its interaction with a large oligomeric protein complex at the sarcolemma and the identicaton of a structurally related protein, utrophin, is leading to the characterization of candidate genes for other neuromuscular disorders

Cystatin C in adipose tissue and stimulation of its production by growth hormone and triiodothyronine in 3T3-L1 cells

Cystatin C (CysC) is a marker for estimation of glomerular filtration rate (GFR). CysC levels may depend not only on clearance/GFR but possibly also on changes in production. Our studies on tissue distribution of CysC protein in mice showed that adipose tissue expresses significant amounts of CysC, suggesting that adipocytes could contribute to circulating CysC levels in vivo. As growth hormone (GH) and triiodothyronine (T) increase both GFR and CysC (increased in acromegaly and hyperthyroidism) in vivo, we studied whether they could increase CysC production in 3T3-L1 adipocytes in vitro. CysC accumulated in culture media of 3T3-L1 adipocytes in a time-dependent fashion. GH and T both (10 nmol/l) increased accumulation of CysC, to 373 ± 14 and 422 ± 20, respectively, vs 298 ± 10 ng per well over 4 days in controls. Thus, GH and T enhance the production of CysC by adipocytes in vitro