Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy.

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G(i)-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G(i)-coupled receptor (Ro1), we provide evidence that increased G(i) signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G(i) signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G(i) signaling in causing cardiac pathology.

The feasibility of a telecommunications service in support of outpatient congestive heart failure care in a diverse patient population.

A home telemonitoring system for patients with congestive heart failure was studied for feasibility and efficacy in a diverse patient population. Fifty patients used the service, in which they weighed themselves and answered yes/no questions about symptoms. Changes in patient weights or symptoms prompted a nurse to call the patient and/or the physician. Patients were given educational and quality of life surveys at enrollment, at 30 days, and at 6 months. The average daily usage rate was 94%. Patients were contacted 57 times--prompting 57 physician notifications, eight medication changes, and 11 nonroutine clinic visits. Patient response to lifestyle surveys showed an improvement in quality of life and improved understanding of prevention measures. Eighty-four percent of patients and 65% of physicians reported satisfaction with the system. This pilot study suggests that home telemonitoring is feasible and has clinical utility in diverse patient groups, and may improve patients' satisfaction and knowledge of self-care. (c)2000 by CHF, Inc.

Benzocaine-induced methemoglobinemia--two case reports related to transesophageal echocardiography premedication.

Benzocaine-induced methemoglobinemia is a potentially life-threatening complication. We report two cases of methemoglobinemia due to topical benzocaine spray used as premedication for transesophageal echocardiography. A high index of suspicion is needed for this readily treatable condition.

Thrombin receptor activating mutations. Alteration of an extracellular agonist recognition domain causes constitutive signaling.

Constitutively active thrombin receptors were generated while constructing chimeric receptors to identify the structural basis for thrombin receptor agonist specificity. Substitution of eight amino acids from the Xenopus receptor's second extracellular loop (XECL2B) for the cognate sequence in the human thrombin receptor was sufficient to confer robust constitutive activity. Smaller substitutions within the XECL2B site yielded less constitutive activation, and substitution of several unrelated sequences at this site caused no activation. Expression of the XECL2B receptor caused high basal 45Ca efflux in Xenopus oocytes and high basal phosphoinositide hydrolysis and reporter gene induction in COS cells. Of note, a mutant receptor in which all four of the Xenopus thrombin receptor's extracellular segments replaced the cognate human sequences showed much less constitutive activity than XECL2B and preserved responsiveness to agonist. This partial complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures suggests that the XECL2B mutation causes constitutive activation by altering interactions among the human receptor's extracellular domains. Thus, a change in an extracellular loop of a G protein-coupled receptor can transmit information across the cell membrane to cause signaling, perhaps via a conformational change similar to that caused by agonist binding. Indeed, the site of the activating mutation in XECL2B coincides with a putative agonist-docking site, supporting the hypothesis that agonist interactions with the thrombin receptor's extracellular loops contribute to receptor activation.

Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site.

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn87 in the Xenopus receptor's amino-terminal exodomain and Glu for Leu260 in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg5a in the human agonist peptide might normally interact with Glu260 in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu260 was converted to Arg. An "arginine scan" of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu260 region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu260 region and to a lesser degree Phe87 as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the "correct" agonist and inhibit responsiveness to an "incorrect" agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.

Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface.

G-protein-coupled receptors for catecholamines and some other small ligands are activated when agonists bind to the transmembrane region of the receptor. The docking interactions through which peptide agonists activate their receptors are less well characterized. The thrombin receptor is a specialized peptide receptor. It is activated by binding its tethered ligand domain, which is unmasked upon receptor cleavage by thrombin. Human and Xenopus thrombin receptor homologues are each selectively activated by the agonist peptide representing their respective tethered ligand domains. Here we identify receptor domains that confer this agonist specificity by replacing the Xenopus receptor's aminoterminal exodomain and three extracellular loops with the corresponding human structures. This switches receptor specificity from Xenopus to human. The specificity of these thrombin receptors for their respective peptide agonists is thus determined by their extracellular surfaces. Our results indicate that agonist interaction with extracellular domains is important for thrombin receptor activation.

Excimer laser ablation of the lens.

Ablation of the bovine crystalline lens was studied using radiation from an excimer laser at four ultraviolet wave lengths as follows: 193 nm (argon fluoride), 248 nm (krypton fluoride), 308 nm (xenon chloride), and 351 nm (xenon fluoride). The ablation process was quantitated by measuring mass ablated with an electronic balance, and characterized by examining ablation craters with scanning electron microscopy. The highest ablation rate was observed at 248 nm with lower rates at 193 and 308 nm. No ablation was observed at 351 nm. Scanning electron microscopy revealed the smoothest craters at 193 nm while at 248 nm there was vacuolization in the crater walls and greater disruption of surrounding tissue. The craters made at 308 nm did not have as smooth a contour as the 193-nm lesions. The spectral absorbance of the bovine lens was calculated at the wavelengths used for ablation and correlated with ablation rates and thresholds. High peak-power, pulsed ultraviolet laser radiation may have a role in surgical removal of the lens.