Biomedical subjects

Jeffrey Robbins

Publications and source records attributed to Jeffrey Robbins.

At least 37 records · Page 2Linked to original sources

PubMed2005

Transgenic rabbit model for human troponin I-based hypertrophic cardiomyopathy.

BACKGROUND: Transgenic and gene-targeted models have focused on the mouse. Fundamental differences between the mouse and human exist in Ca2+ handling during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system. METHODS AND RESULTS: Cardiac troponin I (cTnI) mutations can cause familial hypertrophic cardiomyopathy. An inhibitory domain mutation, arginine146-->glycine (cTnI(146Gly)), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI(146Gly) levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI(146Gly) expression led to a leftward shift in the force-pCa2+ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI(146Gly) myocytes, twitch relaxation amplitudes were smaller than in normal cells, but [Ca]i transients and sarcoplasmic reticulum Ca2+ load were not different. Detrended fluctuation analysis of the QT(max) intervals was used to evaluate the cardiac repolarization phase and showed a significantly higher scaling exponent in the transgenic animals. CONCLUSIONS: Expression of modest amounts of cTnI(146Gly) led to subtle defects without severely affecting cardiac function. Aberrant connexin organization, subtle morphological deficits, and an altered fractal pattern of the repolarization phase of transgenic rabbits, in the absence of entropy or other ECG abnormalities, may indicate an early developing pathology before the onset of more obvious repolarization abnormalities or major alterations in cardiac mechanics.

Animals↗

PubMed2005

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.

Adenoviridae↗

PubMed2005

Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Animals↗

PubMed2005

Ras-related signaling pathways in valve development: ebb and flow.

Congenital heart defects affect approximately 1 in every 100 live births, and deficits in the formation of the mitral, tricuspid, and outflow tract valves account for 20-25% of all cardiac malformations. Mutations in genes that affect Ras signaling have been identified in individuals with congenital valve disease associated with Noonan syndrome and neurofibromatosis type 1. Dissection of Ras-related signaling pathways during valvulogenesis provides seminal insight into cellular and molecular mechanisms that contribute to congenital heart disease.

Animals↗

PubMed2005

Usability factors in the organization and display of disparate information sources in the operative environment.

The integration and presentation of information from a number of disparate sources in the operative environment raises a number of usability and human factors challenges. Through a collaborative effort, a display combining persistent and dynamically switching panes provides a rich source of information to help orient team members, provide indications of case progress, and organize information into stage-based tabbed panes. This provides maximal flexibility within visibility and usability constraints.

Data Display↗

PubMed2005

Integration of hospital information systems, operative and peri-operative information systems, and operative equipment into a single information display.

The integration of disparate information systems in the operative environment allows access to information that is typically unseen or unused. Through a collaborative effort, a variety of information systems and surgical equipment are being integrated. This provides improved context-sensitive information display and decision support and improved access to information to improve workflow, safety and visualization of information that was previously unattainable.

Data Display↗

PubMed2004

Impact of beta-myosin heavy chain expression on cardiac function during stress.

OBJECTIVES: In failing mouse and human hearts, a shift in myosin heavy chain (MHC) isoform content from alpha to beta can occur. However, the impact of this phenomenon on disease progression is not well understood. Therefore, using transgenic (TG) mice, we tested how a pre-existing shift from alpha- to beta-MHC affects cardiac function under chronic mechanical or pharmacologic cardiovascular stress. BACKGROUND: Expression of beta-MHC is considered to be energetically favorable, but this might be offset by depressed cardiac function. METHODS: Transgenic mice with near-complete replacement of the normally predominant alpha- with beta-MHC were subjected to cardiac stress. RESULTS: At baseline, TG mice show moderately reduced cardiac contractile function but are otherwise healthy with normal ventricular morphology. After four weeks of swimming, both TG and non-TG animals showed a 20% increase in left ventricular (LV)/body weight ratios. The TG hearts displayed mildly greater end-diastolic and end-systolic LV diameters than nontransgenic hearts after training, but no signs of LV failure were observed. However, chronic stimulation with isoproterenol resulted in augmented LV hypertrophy with signs of LV decompensation in TG mice. Furthermore, in a post-infarction failure model, TG hearts displayed accelerated LV dilation and a faster decline of shortening fraction. CONCLUSIONS: Expression of beta-MHC appears to be disadvantageous to the mice under severe cardiovascular stress, implying that the alpha-->beta-MHC isoform shift observed in cardiac disease may be a maladaptive response.

Animals↗

PubMed2004

Physiological growth synergizes with pathological genes in experimental cardiomyopathy.

Hundreds of signaling molecules have been assigned critical roles in the pathogenesis of myocardial hypertrophy and heart failure based on cardiac phenotypes from alpha-myosin heavy chain-directed overexpression mice. Because permanent ventricular transgene expression in this system begins during a period of rapid physiological neonatal growth, resulting phenotypes are the combined consequences of transgene effects and normal trophic influences. We used temporally-defined forced gene expression to investigate synergy between postnatal physiological cardiac growth and two functionally divergent cardiomyopathic genes. Phenotype development was compared various times after neonatal (age 2 to 3 days) and adult (age 8 weeks) expression. Proapoptotic Nix caused ventricular dilation and severe contractile depression in neonates, but not adults. Myocardial apoptosis was minimal in adults, but was widespread in neonates, until it spontaneously resolved in adulthood. Unlike normal postnatal cardiac growth, concurrent left ventricular pressure overload hypertrophy did not synergize with Nix expression to cause cardiomyopathy or myocardial apoptosis. Prohypertrophic Galphaq likewise caused eccentric hypertrophy, systolic dysfunction, and pathological gene expression in neonates, but not adults. Thus, normal postnatal cardiac growth can be an essential cofactor in development of genetic cardiomyopathies, and may confound the interpretation of conventional alpha-MHC transgenic phenotypes.

Amino Acid Sequence↗

PubMed2004

In vivo and in vitro analysis of cardiac troponin I phosphorylation.

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.

Animals↗

PubMed2004

Charged residue alterations in the inner-core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility.

Two important charge differences between the alpha- and beta-tropomyosin (TM) isoforms are the exchange of a serine residue in the inner-core region at position 229, and a histidine residue at the carboxy-terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in alpha-TM to their beta-TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and -dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in alpha-TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: alpha-TMSer229Glu and alpha-TMHis276Asn. Molecular analyses show that 98% of native alpha-TM is replaced by mutated protein in alpha-TM229 hearts whereas alpha-TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that alpha-TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and -dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in alpha-TM229 hearts. alpha-TM276 hearts show a decrease only in -dP/dt (14%) and TPP is increased. pCa(2+)-tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in alpha-TM229 but no change in alpha-TM276 preparations. Force-[Ca(2+)](IC) measurements from intact papillary fibres indicate that alpha-TM276 fibres produce more force per given [Ca(2+)](IC) when compared to NTG fibres, while alpha-TM229 fibres produce less force per given [Ca(2+)](IC). These data demonstrate that changing charged residues at either the inner-core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.

Animals↗

PubMed2004

Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction.

ANG II type 2 receptor (AT(2)) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca(2+) concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT(2) was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT(2) in the ventricular myocytes and partial colocalization of AT(2) with AT(1). Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca(2+) concentration, 25 degrees C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca(2+)-dependent contractile dysfunction in myocytes from AT(2) transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT(2) expression. Second, activity of the Na(+)/H(+) exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to beta-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to beta-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca(2+) in wild-type myocytes but caused a negative inotropic effect in myocytes from AT(2) transgenic mice. In contrast with beta-adrenergic response, the depressed response to ANG II was related to level of AT(2) overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT(2) has the potential to cause Ca(2+)- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.

Ammonium Chloride↗

PubMed2004

Desmin-related cardiomyopathy in transgenic mice: a cardiac amyloidosis.

An R120G missense mutation in the small heat shock protein alpha-B-crystallin (CryAB(R120G)) causes desmin-related cardiomyopathy (DRM). DRM is characterized by the formation of aggregates containing CryAB and desmin, and it can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. In this article, we show that expression of CryAB(R120G) leads to the formation of electron-dense bodies characteristic of the DRMs and identify these bodies as aggresomes, which are characteristic of the neurodegenerative diseases. Cardiomyocytes transfected with adenovirus containing CryAB(R120G) establish the necessity and sufficiency of CryAB(R120G) expression for aggresome formation. The commonality of these aggresomes with oligomeric protein aggregates found in the amyloid-related degenerative diseases was corroborated by the presence of high levels of amyloid oligomers that may represent a primary toxic species in the amyloid diseases. These oligomeric amyloid intermediates are present also in cardiomyocytes derived from many human dilated and hypertrophic cardiomyopathies.

Amyloidosis↗

PubMed2004

Regulation of cardiac contractility by Rab4-modulated beta2-adrenergic receptor recycling.

Catecholaminergic activation of myocardial beta-adrenergic receptors (betaAR) is the principle mechanism regulating cardiac function. Agonists desensitize betaAR through G protein-coupled receptor kinase-mediated uncoupling and beta-arrestin-mediated internalization. Although inhibition of myocardial G protein-coupled receptor kinase-2 enhances cardiac function and reverses heart failure, pathophysiological effects of modulated betaAR internalization/recycling are unknown. We used mutation and transgenic expression of Rab4, which regulates vesicular transport of heptahelical receptors to plasma membranes, to interrogate in vivo betaAR trafficking and cardiac function. Expression of constitutively active Rab4 Q72L had no effects on cardiac structure or function, but dominant inhibitor Rab4 S27N impaired responsiveness to endogenous and exogenous catecholamines. To relate betaAR trafficking to diminished cardiac function, Rab4 mutant mice were crossbred with mice overexpressing human beta2AR. In unstimulated beta2AR overexpressors, beta2AR localized to heavier endosomes and translocated to lighter, caveolin-rich fractions after isoproterenol stimulation. Coexpression of beta2AR with activated Rab4 Q72L caused loss of receptors from heavier endosomes while retaining normal inotropy. In contrast, coexpression of beta2AR with inhibitory Rab4 S27N mimicked isoproterenol-induced receptor redistribution to caveolae, with diminished cardiac inotropy. Rab4 inhibition alone prevented resensitization after isoproterenol-induced in vivo adrenergic desensitization. Confocal and ultrastructural analyses revealed bizarre vesicular structures and abnormal accumulation of beta2AR in the sarcoplasm and subsarcollema of Rab4 S27N, but not Q72L, mice. These data provide evidence for constant bidirectional sarcollemal-vesicular betaAR trafficking in the in vivo heart and show that Rab4-mediated recycling of internalized betaAR is necessary for normal cardiac catecholamine responsiveness and resensitization after agonist exposure.

Adrenergic beta-2 Receptor Agonists↗

PubMed2004

Overexpression of phospholamban in slow-twitch skeletal muscle is associated with depressed contractile function and muscle remodeling.

The relative amount of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and its crucial inhibitor phospholamban (PLN) are closely regulated and play a pivotal role in maintaining muscle function. The functional importance of PLN has been intensively investigated in cardiac muscle. However, little is known about the role of PLN in the slow-twitch skeletal muscle, which expresses a significantly lower level of PLN but a similar level of SERCA2a compared with cardiac muscle. Thus, to define the physiological significance of PLN in slow-twitch skeletal muscle, we generated transgenic mice with PLN-specific overexpression in soleus, which is largely composed of slow-muscle fibers. The PLN protein levels and the PLN/SERCA2a ratio in transgenic soleus were comparable with those in cardiac muscle. Assessment of isometric-twitch contractions indicated that PLN overexpression was associated with depressed rates of contraction and relaxation, which were not linked to reduced SERCA2a abundance, although the levels of other key Ca2+-handling proteins, including ryanodine receptor, FKBP12, and L-type Ca2+ channel, were significantly decreased. However, isoproterenol stimulation reversed the inhibitory effects of PLN on the transgenic soleus twitch kinetics. Furthermore, the PLN-overexpressing soleus had smaller muscle size, mass, and cross-sectional area compared with wild-types. Interestingly, the percentage of slow fibers was increased in PLN-overexpressing soleus. Taken together, these findings indicate that increased PLN expression in slow-twitch skeletal muscle is associated with impaired contractile function and muscle remodeling.

Animals↗

PubMed2004

Disruption of Rho signaling results in progressive atrioventricular conduction defects while ventricular function remains preserved.

Recent studies suggest that RhoA and Rac1 mediate hypertrophic signals in cardiac myocyte hypertrophy. However, effects on cardiac function caused by inhibition of their activity in the heart have yet to be evaluated. Cardiac-specific inhibition of Rho family protein activities was achieved by expressing Rho GDIalpha, an endogenous specific GDP dissociation inhibitor for Rho family proteins, using the alpha-myosin heavy-chain promoter. Increased expression of Rho GDIalpha led to atrial arrhythmias and mild ventricular hypertrophy in adult mice (4-7 months). However, left ventricular systolic and diastolic function was largely preserved before and after the development of cardiac hypertrophy, indicating that Rho GTPases are not required to maintain ventricular contractile function under basal physiological condition. Electrocardiography and intracardiac electrophysiological studies revealed first-degree atrioventricular (AV) block in the transgenic heart at 1 week of age, which further progressed into second-degree AV block at 4 weeks of age before the development of cardiac hypertrophy. Expression of connexin 40 dramatically decreased from 1 week to 4 weeks of age in the transgenic heart, which may contribute in part to the conduction defects in the transgenic mice. This study provides novel evidence for an important role of Rho GTPases in regulating AV conduction.

Adaptation, Physiological↗

PubMed2004

PKC-alpha regulates cardiac contractility and propensity toward heart failure.

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Animals↗

PubMed2004

Charged residue changes in the carboxy-terminus of alpha-tropomyosin alter mouse cardiac muscle contractility.

Striated muscle tropomyosin (TM) is an essential thin filament protein that is sterically and allosterically involved in calcium-mediated cardiac contraction. We have previously shown that overexpressing the beta-TM isoform in mouse hearts leads to physiological changes in myocardial relaxation and Ca(2+) handling of myofilaments. Two important charge differences in beta-TM compared to alpha-TM are the exchange of serine and histidine at positions 229 and 276 with glutamic acid and asparagine, respectively, imparting a more negative charge to beta-TM relative to alpha-TM. Our hypothesis is that the net charge at specific sites on TM might be a major determinant of its role in modulating cardiac muscle performance and in regulating Ca(2+) sensitivity of the myofilaments. To address this, we generated transgenic (TG) double mutation mouse lines (alpha-TM DM) expressing mutated alpha-TM at the two residues that differ between alpha- and beta-TM (Ser229Glu + His276Asn). Molecular analyses show 60-88% of the native TM is replaced with alpha-TM DM in the different TG lines. Work-performing heart analyses show that alpha-TM DM mouse hearts exhibit decreased rates of pressure development and relaxation (+dP/dt and -dP/dt). Skinned myofibre preparations from the TG hearts indicate a decrease in calcium sensitivity of steady state force. Protein modelling studies show that these two charge alterations in alpha-TM cause a change in the surface charges of the molecule. Our results provide the first evidence that charge changes at the carboxy-terminal of alpha-TM alter the functional characteristics of the heart at both the whole organ and myofilament levels.

Actin Cytoskeleton↗

PubMed2004

Genetic modification of the heart: exploring necessity and sufficiency in the past 10 years.

It has now been approximately a decade since the directed genetic manipulation of genes responsible for normal and abnormal cardiovascular function began. During the past 10 years, thousands of embryonic stem cell lines and hundreds of unique animals carrying defined mutations in genes that impact on cardiac structure and/or function have been made. Homologous recombination in the murine genome has allowed both gene ablation and directed mutations to be placed precisely, while development of powerful transcriptional cassettes that can be exquisitely controlled has allowed both gain and loss of function approaches to be used in order to study a protein's function, determining both necessity and sufficiency. Thus we can now ablate or augment expression of a engineered protein in the heart and are able to effectively remodel the cardiac protein profile and study the consequences of a single genetic manipulation at the molecular, biochemical, cytological and physiologic levels. The ability to effectively manipulate cardiac gene expression via transgenesis or gene targeting enables the investigator to extend correlations to defined mechanisms. This review will revisit these basic technologies, outline the strengths and weaknesses of each, and discuss the advances that are on the experimental horizon.

Animals↗