Paul E. Wolkowicz

MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKCε proteolysis in reperfused ferret hearts

Objectives : This paper tests the hypothesis that calpains are activated in the ischemic (I)/reperfused (R) heart and contribute to myocardial stunning. Methods : Isolated ferret hearts were Langendorff perfused isovolumically, and subjected to 20 min of global I followed by 30 min of R in the presence or absence of 0.2 μ M MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa2+-force curves obtained. Samples of left ventricle were extracted, subjected to SDS-PAGE, and Western analyzed for PKCe and PKMe. Results : Perfused ferret hearts exhibit a 43% decline in left ventricular developed pressure during R. Pre-treatment of hearts with MDL-28170 prior to I significantly improves function during R. Trabecular myofilaments from normal hearts have a KD for Ca2+ of 6.27±0.06; I/R decreased the KD to 6.09±0.04; trabeculae from I/R hearts pre-treated with MDL-28170 have a KD of 6.28±0.04. Western analysis shows ferret hearts to contain a single ≈96 kDa species of PKCe. I/R hearts contain the native PKCe and a ≈25 kDa smaller species of PKCe, which corresponds to PKMe, the calpain proteolyzed form of PKCe. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKMe in reperfused hearts. Conclusions : Mechanical stunning during R is sensitive to MDL-28170. Depressed mechanical function is reflected in a hyposensitization of trabecular myofilaments to Ca2+. Western analysis shows that PKMe is present in R hearts.

1H NMR Spectroscopic Imaging of Myocardial Triglycerides in Excised Dog Hearts Subjected to 24 Hours of Coronary Occlusion

BACKGROUND Myocardial ischemic insult causes depression of fatty-acid beta-oxidation and increased fatty-acid esterification with triglyceride (TG) accumulation. This accumulation has been demonstrated to occur in the territory with diminished blood flow surrounding an infarct, ie, the region at risk. To evaluate whether the extent of TG accumulation in the canine heart after 24 hours of ischemia could be detected, we applied myocardial 1H nuclear magnetic resonance (NMR) spectroscopic imaging (SI). METHODS AND RESULTS Seven adult mongrel dogs underwent 24 hours of left anterior descending coronary artery occlusion. Postmortem, the hearts were excised and the size and location of the infarct were determined. With a Philips 1.5-T clinical NMR imaging/spectroscopic system, two-dimensional (2D) 1H NMR SI was performed. TG 1H NMR chemical shift images were reconstructed from the frequency domain spectra by numerical integration. A statistically significant (P < .05) increase in TG signal intensity was demonstrated in the region at risk compared with the nonischemic control region. There was an intermediate quantity of TG in the infarct region. Biochemical determination of tissue TG content (milligrams per gram wet weight) in the control, at-risk, and infarct regions confirmed the 1H NMR measurements. Histological evaluation with oil red O staining also demonstrated graded TG accumulation in myocytes. The highest TG levels were found in the at-risk region and the lowest levels in the control region. CONCLUSIONS By use of 2D 1H NMR SI, the present study confirms and extends previous work that demonstrates preferential accumulation of TG in the reversibly injured myocardium after 24 hours of coronary occlusion. This study provides an important step toward the clinical application of TG imaging. When TG imaging is ultimately possible, resultant data would have diagnostic, prognostic, and therapeutic implications.

Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient (Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

Pharmacological evidence for Orai channel activation as a source of cardiac abnormal automaticity.

Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 μM in left atria and 6 ± 1.6 μM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle.

1H-NMR spectroscopy can accurately quantitate the lipolysis and oxidation of cardiac triacylglycerols

Triacylglycerol metabolism in isolated, perfused hearts from rats fed a diet containing 20% rapeseed oil (RSO) was studied using 1H-NMR spectroscopy. RSO-induced elevation in cardiac triacylglycerols is associated with an increase in the peak area of fatty acid 1H-NMR resonances. The ratio of methyl, gamma-methylene or methylene protons adjacent to a carbon-carbon double bond to the number of methylene protons in these hearts measured by 1H-NMR spectroscopy gives values similar to those derived from previously reported chemical analyses. In addition, the triacylglycerol content of these hearts determined by chemical analysis directly correlates with their content of 1H-NMR visible fatty acid resonances. This quantitative relationship allows the real-time measurement of the rates of cardiac triacylglycerol lipolysis using 1H-NMR spectroscopy. Rates of triacylglycerol lipolysis measured using 1H-NMR spectroscopy are similar to those previously measured by chemical methods. Triacylglycerol lipolysis measured using 1H-NMR spectroscopy occurs at a significantly faster rate in hearts perfused in the presence or absence of glucose when compared to hearts perfused with glucose and acetate or medium-chain fatty acids. Finally, the rate of triacylglycerol lipolysis in glucose perfused hearts is linearly related to work output. These results demonstrate that 1H-NMR spectroscopy can accurately quantitate triacylglycerol content and metabolism in the rapeseed oil-fed rat model. 1H-NMR spectroscopic or imaging techniques may be useful in the real-time evaluation of cardiac triacylglycerol content and metabolism.

Evidence that 2-aminoethoxydiphenyl borate provokes fibrillation in perfused rat hearts via voltage-independent calcium channels.

We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 μM 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 μM 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 μM 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 μM 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.

Occupation of the prostaglandin E2-type 1 receptor increases rat atrial contractility via a Y-27632-sensitive pathway.

This study investigated whether rat left atria (LA) contain the prostaglandin E2 type 1 receptor (EP1) and whether EP1 occupation induces positive inotropic responses in superfused LA. Western analysis demonstrated that LA contain EP1 and the EP1 splice variant. Exposing isolated, superfused LA to 17-phenyl trinor PGE2, an EP1 agonist, increased isometric contractile force and its corresponding dF/dTs to approximately 70% of the isoproterenol maximum with an EC50 of approximately 80 nM. In contrast, agonists for EP2, EP3, and EP4 caused little change in LA function. While the EP1 antagonists SC-51089 and SC-19220 blocked 17-phenyl trinor PGE2-induced inotropy, neither prazosin, nadolol, atropine nor EI-283, a pan-specific protein kinase C inhibitor, affected 17-phenyl trinor PGE2-induced inotropy. However, Y-27632 and HA-1077, inhibitors of rho A-activated protein kinases, prevented and reversed the increase in LA contractility that occurred in the presence of 17-phenyl trinor PGE2. Thus, atria contain EP1 and EP1 occupation increases LA contractility via a pathway sensitive to inhibitors of rho A-activated protein kinases.

2-APB induces instability in rat left atrial mechanical activity.

Atrial contractile abnormalities are common clinical disorders but few pharmacological models can reliably produce such abnormalities in isolated atrial muscle. Since sarcoplasmic reticulum (SR) calcium leak may underlie these contractile irregularities, we investigated whether 2-aminoethoxydiphenyl borate (2-APB), a calcium leak-inducer, affects mechanical function in isolated, superfused rat left atria. Exposing left atria paced at 3 Hz to >10 μM 2-APB produced sporadic mechanical events that occurred in the absence of pacing stimulus. Prolonging atrial diastole in the presence of 2-APB produced spontaneous mechanical activity (SMA) defined as numerous mechanical events occurring in the absence of pacing stimulus. SMA depends on atrial sodium and chloride gradients as decreasing superfusate concentration of either ion suppressed SMA. Increasing superfusate potassium to produce an EK of ∼−74mV reversed SMA, revealing possible membrane potential sensitivity. Mechanical function decreased with time in left atria treated with 2-APB and low sodium or the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) compared with atria exposed to low sodium or DIDS alone, suggesting 2-APB may decrease left atrial SR activator calcium. Thus, 2-APB produces instability in regular left atrial mechanical activity that may require forward-mode sodium-calcium exchange and chloride channel activities. This data identify a new model for studying atrial contractile abnormalities.

Stereoselective synthesis of a conformationally defined cyclohexyl carnitine analogue that binds CPT-1 with high affinity

Carnitine (1, 3-hydroxy-4-trimethylammoniobutyrate) is important in mammalian tissue as a carrier of acyl groups. In order to explore the binding requirements of the carnitine acyltransferases for carnitine, we designed conformationally defined cyclohexyl carnitine analogues. These diastereomers contain the required gauche conformation between the trimethylammonium and hydroxy groups but vary the conformation between the hydroxy and carboxylic acid groups. Here we describe the synthesis and biological activity of the all-trans diastereomer (2), which was prepared by the ring opening of trans-methyl 2,3-epoxycylohexanecarboxylate with NaN3. Racemic 2 was a competitive inhibitor of neonatal rat cardiac myocyte CPT-1 (K(i) 0.5 mM for racemic 2; K(m) 0.2 mM for L-carnitine) and a noncompetitive inhibitor of neonatal rat cardiac myocyte CPT-2 (K(i) 0.67 mM). These results suggest that 2 represents the bound conformation of carnitine for CPT-1.

N-cadherin coordinates AMP kinase-mediated lung vascular repair.

Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherins role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherins interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherins intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.