Showing papers by "Jennifer E. Van Eyk published in 2002"

Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane.

Abstract: Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K+) channel, the mitochondrial adenosine triphosphate–sensitive K+ channel (mitoKATP), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K+ channel was found on the mitochondrial inner membrane (mitoKCa) of guinea pig ventricular cells. MitoKCa significantly contributed to mitochondrial K+ uptake of the myocyte, and an opener of mitoKCa protected hearts against infarction.

p21-Activated Kinase Increases the Calcium Sensitivity of Rat Triton-Skinned Cardiac Muscle Fiber Bundles via a Mechanism Potentially Involving Novel Phosphorylation of Troponin I

Abstract: Phosphorylation of myofilament proteins by kinases such as cAMP-dependent protein kinase and protein kinase C has been shown to lead to altered thin-filament protein-protein interactions and modulation of cardiac function in vitro. In the present study, we report that a small GTPase-dependent kinase, p21-activated kinase (PAK), increases the calcium sensitivity of Triton-skinned cardiac muscle fiber bundles. Constitutively active PAK3 caused an average 1.25-fold (25.0±6.0%, n=6) increase in force at pCa 5.75, 1.44-fold (44.0±7.78%, n=6) at pCa 6.25, and 2.41-fold (141.2±23.7%, n=4) at pCa 6.5, representing a change in pCa 50 value of approximately 0.25. Constitutively active PAK3 produced no change in force under conditions of relaxation (pCa 8.0) or maximal contraction (pCa 4.5). Furthermore, an inactive, kinase-dead form of PAK3 failed to produce any change in force development at any pCa value. The myofilament proteins phosphorylated by PAK3, at pCa 6.5, are desmin, troponin T, troponin I, and an unidentified 70-kDa protein. Importantly, cardiac troponin I was found to be phosphorylated at serine 149 of human cardiac troponin I, representing a novel phosphorylation site. These findings suggest a novel mechanism of modulating the calcium sensitivity of cardiac muscle contraction.

Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle.

Abstract: The application of protein separation methodologies, such as reversed phase chromatography, should allow differential separation of the proteome, or at least specific subproteomes, comparable to that achieved by two-dimensional electrophoresis (2-DE). A rapid sequential protein extraction method (termed "IN Sequence") was developed to isolate three distinct subproteomes of cardiac muscle. Two subproteomes, those enriched for the cytoplasmic or myofilament proteins, can be separated by either reversed phase high performance liquid chromatography (RP-HPLC) or 2-DE. Reversed phase HPLC of the myofilament protein enriched extract was optimized for resolution and peak numbers by altering flow rate, gradient rate and the organic modifiers, isopropanol and acetronitrile. The myofilament protein enriched extract from failing swine heart, due to coronary artery ligation (LAD), was compared to the extract from a sham operated animal (SHAM). The HPLC chromatograms of these extracts were similar, but distinctive in many regions. The HPLC fractions, collected within some of these distinct regions of the chromatograms were analyzed using peptide mass fingerprinting - mass spectrometry and immunoblot analysis. Two myofilament proteins, troponin T and myosin heavy chain, were identified and found differentially modified in the SHAM and LAD hearts. Both troponin T and myosin heavy chain are problematic proteins for 2-DE, but yet they were resolved by reversed phase chromatography. Therefore, RP-HPLC can be used in conjunction with 2-DE to enhance protein separation of myofilament protein subproteome.

Differential Detection of Skeletal Troponin I Isoforms in Serum of a Patient with Rhabdomyolysis: Markers of Muscle Injury?

Abstract: Unlike the extensive research devoted to the development of troponin-based diagnostic assays for myocardial disease, much less effort has been expended on the development of a counterpart for skeletal muscle disorders. The consensus that the cardiac troponins [cardiac troponin I (cTnI) and/or cTnT] should be used for diagnosis of myocardial infarction (MI), as well as for diagnosis and management of unstable angina, is based on their superior tissue specificity over such conventional markers as creatine kinase [(CK); see Refs. (1)(2)]. Although CK is the most common serum marker for skeletal muscle injury, it is not ideal for several reasons, including lack of tissue specificity, inability to reveal damage to specific skeletal fiber types (fast or slow), and inappropriately low values when glutathione concentrations are decreased because of liver or multiple-organ failure (3). Skeletal troponin I (sTnI), with its two distinct isoforms [fast (fsTnI) and slow (ssTnI)], like cTnI and cTnT, may have a similar advantage over conventional markers for detecting skeletal-muscle injury. In 1996, Rama et al. (4) described an experimental immunoenzymatic assay for sTnI using antibodies that cross-react with both sTnI and cTnI. The assumption of the investigators was that the concentrations of cTnI in patients with skeletal injury would be negligible. Others have since used this assay (5)(6)(7). For example, Onuoha et al. (7) found that serum sTnI reflects the severity and type of orthopedic and soft tissue injury. However, because this assay does not differentiate between the two isoforms of sTnI, which have a sequence homology of ∼56%, information about selective damage to particular fiber types is unavailable. Posttranslational modifications to the analyte, such as degradation, are also undetected by this assay. We applied our Western blot–direct serum analysis (WB-DSA) procedure (8), originally developed and successfully used for the detection …

Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T.

Abstract: Proteomics strongly relies on the separation of complex protein mixtures, with two-dimensional electrophoresis (2-DE) being a commonly used technique. However efficient separation requires adequate solubilization of the original sample which will determine whether all proteins are accurately represented. Cardiac muscle has presented a particular challenge to solubilization. Here we have optimized the solubilization, separation and detection of the myofilament protein troponin T (TnT). Human left ventricular tissue from a rejected donor transplant heart was homogenized under 19 different conditions and subjected to 2-DE and Western blot analysis for TnT. The optimal conditions for isoelectric focusing of intact TnT requires homogenization in 6 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, with the addition of NaCl (2.5 M final concentration). TnT degradation products present in this severely damaged heart however, were differentially extracted from both each other and the intact molecule under the various conditions used. Despite adequate focusing of TnT it was found that a nonglutaraldehyde silver staining protocol, that is compatible with subsequent mass spectrometry, has greatly reduced sensitivity for TnT compared to Coomassie blue. Thus, care is required to avoid misrepresentation of troponin T in proteomic analysis in cardiac tissue.

Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie, silver, mass spectrometry, and Western blotting.

Abstract: Proteomic analysis of myocardial tissue from patient populations is critical to our understanding of cardiac disease, but has been limited until now by the small size of biopsies (approximately 20-50 microg), and complicated by the difference in relative abundance of contractile proteins over other cellular components. Here we describe an approach to analysis of myocardial biopsies from patients undergoing coronary artery bypass surgery. First, individual biopsies are selectively extracted, producing subfractions that correspond to the contractile proteins and the cytosolic proteins. Two-dimensional electrophoresis separated proteins are detected by first staining with Coomassie blue then silver, to permit a wider range of accurate quantification. Western blotting of two-dimensional separated samples, to validate peptide mass fingerprinting data, previously required additional gel separations for transfer since staining protocols are not compatible with transfer to membranes or immunoblotting. An existing silver destaining protocol was adapted to allow removal of silver from a whole gel, followed by transfer and Western blotting. An existing Coomassie blue removal protocol was also adapted to permit Western blotting of gels stained with Coomassie blue and silver. Together, these techniques permit peptide mass fingerprinting concurrent with Western blotting of a single protein spot from a single biopsy, eliminating the need for repeated gel separations, and improving spot alignment between immunoblots and stained gels. In the end, this approach may allow a more complete characterization of protein changes in small human biopsies, and also reduce the number of repeated gel separations necessary for a standard proteomic analysis.

Detection of cardiac troponin I early after onset of chest pain in six patients.

Abstract: Patients presenting to the emergency departments (ED) with symptoms of acute coronary syndrome (ACS) and with a nondiagnostic electrocardiogram (ECG) pose a management challenge (1). Cardiac troponins [(cTns), tropinin I (cTnI) and tropinin T [cTnT)], creatine kinase (CK), and CK-MB are frequently used in the assessment of ACS. cTns are superior in their analytical specificity and diagnostic sensitivity and specificity for myocardial injury (2)(3). Findings from both animal and clinical studies show that cTnI is released into the blood in various cardiac conditions, including angina, acute myocardial infarction (1)(4)(5), congestive heart failure (6), and myocarditis (7). Because cTns in serum represent myocardial damage and increased risk of future adverse outcomes (8), improving the detection of serum cTns has implications for better diagnosis of myocardial damage and better risk stratification for patients with ACS. With current clinical assays, cTns are detectable in the circulation 4–6 h after the onset of pain in acute myocardial infarction, peaking within 12–24 h and remaining increased for a few days (9). However, a recently developed Western blot method, WB-DSA (10), detected minute amounts of cTnI in serum of patients undergoing bypass surgery within 10 min after reperfusion (11), suggesting increased detection of TnI by the WB-DSA method. Although WB-DSA does not permit analysis of troponin’s quaternary structure, it does allow accurate assessment of the chemical status of individual troponin subunits, such as the extent and pattern of cTnI degradation. cTnI is specifically degraded in ischemic/reperfused injured rat myocardium (4)(12), and TnI degradation products are detected in myocardium of patients undergoing coronary artery bypass surgery. Because ACS represents a spectrum of cardiac pathophysiology, unique patterns of cTnI degradation may be present in serum at various points along this spectrum and detectable by the WB-DSA. This study presents a …