Pleiotropic Responses Research Articles

Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Lysophosphatidic acid mediates pleiotropic responses in skeletal muscle cells

Lysophosphatidic acid (LPA) is a potent modulator of growth, cell survival, and apoptosis. Although all four LPA receptors are expressed in skeletal muscle, very little is known regarding the role they play in this tissue. We used RT-PCR to demonstrate that cultured skeletal muscle C2C12 cells endogenously express multiple LPA receptor subtypes. The demonstration that LPA mediates the activation of ERK1/2 MAP kinase and Akt/PKB in C2C12 cells is consistent with the widely observed mitogenic properties of LPA. In spite of these observations, LPA did not induce proliferation in C2C12 cells. Paradoxically, we found that prolonged treatment of C2C12 cells with LPA led to caspase 3 and PARP cleavage as well as the activation of stress-associated MAP kinases JNK and p38. In spite of these typically pro-apoptotic responses, LPA did not induce cell death. Blocking ERK1/2 and Akt/PKB activation with specific pharmacological inhibitors, nevertheless, stimulated LPA-mediated apoptosis. Taken together, these results suggest that both mitogenic and apoptotic responses serve to counterbalance the effects of LPA in cultured C2C12 cells.

Engaging the vascular component of the tumor response

Recent research has shed new light on the critical role of tissue microvasculature in regulating the tumor response to radiation and drugs. In this issue of Cancer Cell, Moeller et al.(2005) demonstrate that HIF-1 activation during the course of fractionated radiotherapy initiates pleiotropic adaptive responses in both tumor cells and the microvascular network, radiosensitizing tumor cells but concomitantly conferring tumor radioresistance due to protection of the microvascular endothelium. HIF-1 thus serves as a legitimate target for differential modulation of tissue response to radiation.

Human VIP-α: an emerging biologic response modifier to treat primary pulmonary hypertension

Primary pulmonary hypertension (PPH) is a rare life-threatening disorder of unknown etiology manifested by chronic elevation of pulmonary arterial pressure. Given that pulmonary vasoconstriction, endothelial and vascular smooth muscle cell proliferation and in situ thrombosis contribute appreciably to the evolution of PPH, treatment with vasodilators, antiproliferative drugs and anticoagulants, alone or in combination, constitute the pharmacologic standard of care. To this end, long-term administration of oral calcium channel blockers, prostacyclin analogs by various routes and oral endothelin-1 receptor antagonists, alone or in combination, is efficacious in treating patients with PPH. Unfortunately, efficacy is hampered by poor stability, delivery and bioavailability, and by systemic toxicity. Hence, there is an ongoing need to develop and test new drugs to treat patients with PPH. To address this issue, a novel, targeted, long-acting, biocompatible and safe sterically stabilized liposomal and micellar formulation of human vasoactive intestinal peptide (VIP) was developed and tested for human use: the 28-amino acid pleiotropic biologic response modifier, human VIP-α. The long-lasting salutary effects of phospholipid-associated VIP on vasomotor tone and arterial pressure were expressed at low concentrations solely in diseased animals and were independent of its route of administration. Thus, the author proposes that human VIP-α could be developed as a safe long-acting drug to treat patients with PPH.

Tissue expression of alternatively spliced GFRα1, NCAM and RET isoforms and the distinct functional consequence of ligand-induced activation of GFRα1 isoforms

Glial-cell-line-derived neurotrophic factor (GDNF) exerts its effect through a multi-component receptor system consisting of GFRα1, RET and NCAM. Two highly homologous alternatively spliced GFRα1 isoforms (GFRα1a and GFRα1b) have previously been identified. In this study, isoform specific real-time PCR assays were used to quantify the expression levels of GFRα1, RET and NCAM isoforms in murine embryonic and adult tissues. The expression levels of GFRα1b were found to be comparable to that of GFRα1a in peripheral tissues. However, GFRα1a was the predominant isoform expressed in the whole brain. The co-expressions of GFRα1 and the co-receptors were developmentally regulated and differentially expressed in some tissues. Microarray analyses of GFRα1 isoforms transfected cells stimulated with NTN showed distinct and non-overlapping gene profiles. These observations are consistent with the emerging view that the combinatorial interactions of the spliced isoforms of GFRα, RET and NCAM may contribute to the pleiotropic biological responses.

Acute leptin deficiency, leptin resistance, and the physiologic response to leptin withdrawal.

Food restriction and weight loss result in reduced plasma leptin, which is associated with a pleiotropic biologic response. However, because weight loss itself is also associated with changes in numerous other humoral and metabolic signals, it can be difficult to determine the precise features of the biologic response to acute leptin deficiency. To study this response in the absence of changes in nutritional state, we have developed a protocol that allows such analysis in normal, non-food-restricted animals. Wild-type mice are treated with high-dose leptin until fat mass is depleted and, as a consequence, endogenous leptin production is reduced. At this point, exogenous leptin is abruptly withdrawn, thus inducing a state of leptin deficiency in otherwise normal mice. Leptin deficiency is sustained by feeding the animals only as much as they consumed voluntarily before leptin withdrawal. The biologic response to leptin deficiency induced in this manner includes altered neuropeptide levels, decreased energy expenditure, and impaired reproductive and immune function. Replacement of leptin at physiological concentrations after withdrawal of high-dosage leptin blunts, but does not completely block, the hyperphagia and weight regain caused by acute leptin deficiency, nor does it correct the resulting reproductive and immune dysfunction. This suggests that high-dosage leptin treatment induces a state of partial leptin resistance. In aggregate, these studies establish the role of acute hypoleptinemia in regulating energy balance, the immune system, and reproductive function, and further suggest that high-dosage leptin treatment can induce a state of acquired leptin resistance.

Metabolite fingerprinting in transgenic lettuce

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.

AMP-activated Protein Kinase Mediates Phenobarbital Induction of CYP2B Gene Expression in Hepatocytes and a Newly Derived Human Hepatoma Cell Line

Phenobarbital (PB) administration is known to trigger pleiotropic responses, including liver hypertrophy, tumor promotion, and induction of genes encoding drug-metabolizing enzymes. The induction of human CYP2B6 and the rat (CYP2B1) and mouse (Cyp2b10) homologues by PB is mediated by the nuclear receptor constitutive androstane receptor (CAR). The study of CYP2B gene regulation and CAR activity by PB has been difficult due to the lack of a cellular model. In this study, we describe a novel differentiated human hepatoma cell line (WGA), derived from HepG2, which expresses CYP2B6 and CAR. WGA cells represent a powerful system to study the regulation of CYP2B6 gene expression by PB. There is evidence that CAR activity is regulated by phosphorylation and that regulation of some CYP genes depends on the nutritional status of cells. The AMP-activated protein kinase (AMPK) functions as an energy sensor and is activated when cells experience energy-depleting stresses. In this report, we show that addition of 5-amino-imidazole carboxamide riboside, an AMPK activator, to WGA and human hepatocytes induces CYP2B6 gene expression. Expression of a constitutively active form of AMPK mimics the PB induction of CYP2B6 and CYP2B1 gene expression. Conversely, the expression of a dominant negative form of AMPK inhibits the induction of these genes by PB. Finally, we demonstrate, for the first time, that AMPK activity increases in cells cultured with PB. Our data strongly support a role for AMPK in the PB induction of CYP2B gene expression and provide new insights into the regulation of gene expression by barbiturate drugs.

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cγ, STAT1, Gab1 and FRS2α leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2α and FRS2β are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.

Type I interferons and the innate immune response—more than just antiviral cytokines

The role of type I interferon (referred to as IFN in this review) in early antiviral immunity is well known. More recently IFN has been shown to be a potent regulator of adaptive immunity. It is now becoming clear that a broad range of viruses, bacteria and even parasites express ligands capable of stimulating a growing number of signalling pathways that results in, often subtype specific, induction of IFN. Of particular interest are the signalling pathways associated with the Toll-like receptors. This family of receptors, each able to induce signals in response to a variety of ligands, initiates the pro-inflammatory response. They also contain members that have the capacity to induce IFN, making use of, and perhaps promoting the evolution of its pleiotropic responses. Greater knowledge of the events that result in induction of IFN is necessary in understanding the specificity of expression of an increasingly complex and important aspect of our immune system. This may reveal to us further therapeutic opportunities, either in the use of IFN or in the manipulation of their expression. This review details the established knowledge and recent advances made in understanding how and under what circumstances the IFNs are expressed, starting with brief overviews of IFN and Toll-like receptors before following the molecular processes from induction of IFN, activation of the JAK-STAT pathway and finally the expression of interferon stimulated genes and their functions.

Signals for Defense and Growth

Ethylene is a plant hormone that regulates multiple processes, including plant growth and pathogen defense. Dong et al. studied the response of Arabidopsis to exposure to harpin, a protein produced by certain plant pathogenic bacteria that causes pleiotropic responses. In Arabidopsis , harpin causes root elongation, increases in plant size and mass, and resistance to aphid infestation. Using reverse transcription polymerase chain reaction (RT-PCR), the authors demonstrated that many of the genes induced by harpin were also induced by ethylene. Analysis of mutant plants further demonstrated that ethylene signaling, but not salicylic acid or jasmonate signaling, was required for the harpin response. Finally, analysis of plants with mutations in genes encoding two components of the ethylene pathway, EIN2 or EIN5, indicated that EIN2 was required for aphid resistance, whereas EIN5 was required for plant growth enhancement. These results provide insight into the molecular mechanisms by which ethylene can control diverse plant responses. H.-P. Dong. J. Peng, Z. Bao, X. Meng, J. M. Bonasera, G. Chen, S. V. Beer, H. Dong, Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense. Plant Phys. 136 , 3628-3638 (2004). [Abstract] [Full Text]

The interplay between Src and integrins in normal and tumor biology.

Src family nonreceptor protein tyrosine kinases transduce signals that control normal cellular processes such as cell proliferation, adhesion and motility. Normally, cellular Src is held in an inactive state, but in several cancer types, abnormal events lead to elevated kinase activity of the protein and cause pleiotropic cellular responses inducing transformation and metastasis. A prerequisite of the ability of a cancer cell to undergo metastasis into distant tissues is to penetrate surrounding extracellular matrices. These processes are facilitated by the integrin family of cell adhesion molecules. As is the case with Src, altered integrin activity or substrate affinity can contribute to the neoplastic phenotype. Therefore, understanding the interplay between Src and integrin function has been of intense interest over the past few years. This review focuses on the role of Src and integrin signaling in normal cells and how this is deregulated in human cancer. We will identify the key players in the integrin-mediated signaling pathways involved in cell motility and apoptosis, such as FAK, paxillin and p130(CAS), and discuss how Src signaling affects the formation of focal adhesions and the extracellular matrix.

Complex Interplay Among Regulators of Drug Resistance Genes in Saccharomyces cerevisiae

The Gal4p family of yeast zinc cluster proteins comprises regulators of multidrug resistance genes. For example, Pdr1p and Pdr3p bind as homo- or heterodimers to pleiotropic drug response elements (PDREs) found in promoters of target genes. Other zinc cluster activators of multidrug resistance genes include Stb5p and Yrr1p. To better understand the interplay among these activators, we have performed native co-immunoprecipitation experiments using strains expressing tagged zinc cluster proteins from their natural chromosomal locations. Interestingly, Stb5p is found predominantly as a Pdr1p heterodimer and shows little homodimerization. No interactions of Stb5p with Pdr3p or Yrr1p could be detected in our assays. In contrast to Stb5p, Yrr1p is only detected as a homodimer. Similar results were obtained using glutathione S-transferase pull-down assays. Importantly, the purified DNA binding domains of Stb5p and Pdr1p bound to a PDRE as heterodimers in vitro. These results suggest that the DNA binding domains of Pdr1p and Stb5p are sufficient for heterodimerization. Our data demonstrate a complex interplay among these activators and suggest that Pdr1p is a master drug regulator involved in recruiting other zinc cluster proteins to fine tune the regulation of multidrug resistance genes.

Mini ReviewSignalling by the Type 1 Insulin-like Growth Factor Receptor: Interplay with the Epidermal Growth Factor Receptor

The type 1 insulin-like growth factor receptor (IGF-1R) plays an essential role in mammalian growth and development, and has emerged as a candidate therapeutic target in the treatment of cancer. While the pleiotropic cellular responses elicited following tyrosine phosphorylation of the IGF-1R is usually seen to involve the direct recruitment/activation of classical intracellular effector proteins, it is now clear that cross-talk between the IGF-1R and members of distinct receptor families also plays a significant role in effecting intracellular signalling. In recent years, a number of studies have highlighted the interaction(s) between the IGF-1R and the epidermal growth factor receptor (EGFR), another transmembrane tyrosine kinase that is an established cancer target. This review describes the components of the IGF signalling system and gives an overview of the emerging picture of the interrelationship that is now known to exist between the IGF and EGF receptors.

Pretreatment evaluation of combined HIF-1alpha, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer.

Tumor hypoxia has been known to be associated with resistance to radiation and chemotherapy (CRT). Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor induced by hypoxic condition, plays a major role in the pleiotropic response observed under hypoxic conditions. It encodes proteins that play key roles in critical development and physiologic processes, including angiogenesis, glucose transport and erythropoiesis. On the other hand, cell cycle- and apoptosis-control genes p53 and p21 may play major roles in the tumor response to cytotoxic agents such as radiation and chemotherapy. Previous reports have suggested that the regulation of p53 and p21 is HIF-1-dependent. Our aim was to evaluate the expression of the HIF-1alpha, p53 and p21 proteins by immunohistochemistry in biopsy specimens of esophageal squamous cell carcinoma, which were obtained endoscopically from 65 patients before CRT, and then determine whether the levels of expression of these proteins predicted the clinical effectiveness of CRT in individual cancers. Also, to assess the relationship between expression of these proteins and cell death and cellular proliferation activity, we evaluated Ki67 expression and the apoptosis index (TUNEL). HIF-1alpha expression in esophageal cancer was significantly and negatively related to the response to CRT, independently of p53 and p21 expression. Interestingly, 44.4% (12/27) of the HIF-1alpha-negative group showed a complete response to therapy. There was no significant correlation between the expression of HIF-1alpha, p53 and p21 and proliferation and apoptosis. HIF-1alpha overexpression may predict resistance to CRT and may be a helpful guide in choosing between therapeutic strategies, such as intensive combined modality therapy vs. palliative therapy. Combined immunohistochemical evaluation of HIF-1alpha, p53 and p21 protein expression at the pretreatment biopsy is a very useful and powerful indicator of sensitivity to CRT in human esophageal cancer. Our data also indicate the importance of having a clear grasp of the degree of hypoxia (HIF-1alpha) of a tumor, rather than its cellular character (proliferation and apoptosis), to indicate the likely impact of CRT.

Synleurin, a novel leucine-rich repeat protein that increases the intensity of pleiotropic cytokine responses

We have identified and characterized a novel single span transmembrane leucine-rich repeat protein, synleurin, that renders cells highly sensitive to the activation by cytokines and lipopolysaccharide (LPS). The major part of the extracellular domain consists of a leucine-rich repeats (LRR) cassette. The LRR central core has 12 analogous LRR repeating modules arranged in a seamless tandem array. The LRRs are most homologous to that of chondroadherin, insulin-like growth factor binding proteins, platelet glycoprotein V, slits, and toll-like receptors. Synleurin expression was detected at low levels in many tissues, including smooth muscle, brain, uterus, pancreas, cartilage, adipose, spleen, and testis. When synleurin is ecotopically expressed in transfected cells, the cells exhibit amplified responses to bFGF, EGF, PDGF-B, IGF-1, IGF-2, and LPS. Synleurin gene ( slrn) maps to human chromosome at 5q12. The name synleurin reflects its synergistic effect on cytokine stimulation and its prominent leucine-rich repeats.

Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses.

The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathway to trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-beta dependent Ca(2+) flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G alpha(i) association with the receptor is dependent on JAK activation, and the chemokine-mediated Ca(2+) flux that requires phospholipase C-beta activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways--G proteins and tyrosine kinases--in a network that may be essential for mediating their pleiotropic responses.

Interaction of Nitric Oxide with Human Heme Oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization.

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.

Myofilament remodeling during the progression of heart failure

Background: Myocardial failure is associated with complex, pleiotropic responses involving multiple signaling mechanisms and alterations in structure-function relationships. Myofilament proteins are a central component of these responses and may be modified in various ways, including isoform variation, phosphorylation, and degradation. Methods and Results: We have modeled the transition from compensated hypertrophy to failure using a combination of isolated heart and in vitro motility methods in the Dahl salt sensitive rat model. Conclusions: Our results and prior data indicate that myosin isoform switching probably does not have major functional consequences in failing human myocardium. In contrast, a defect in the thin filament appears to play an important role in the Dahl rat and possibly in patients. The mechanism is uncertain, but preliminary data suggest altered phosphorylation of troponin I and/or T.