René J. P. Musters

Diastolic Stiffness of the Failing Diabetic Heart Importance of Fibrosis, Advanced Glycation End Products, and Myocyte Resting Tension

Background— Excessive diastolic left ventricular stiffness is an important contributor to heart failure in patients with diabetes mellitus. Diabetes is presumed to increase stiffness through myocardial deposition of collagen and advanced glycation end products (AGEs). Cardiomyocyte resting tension also elevates stiffness, especially in heart failure with normal left ventricular ejection fraction (LVEF). The contribution to diastolic stiffness of fibrosis, AGEs, and cardiomyocyte resting tension was assessed in diabetic heart failure patients with normal or reduced LVEF. Methods and Results— Left ventricular endomyocardial biopsy samples were procured in 28 patients with normal LVEF and 36 patients with reduced LVEF, all without coronary artery disease. Sixteen patients with normal LVEF and 10 with reduced LVEF had diabetes mellitus. Biopsy samples were used for quantification of collagen and AGEs and for isolation of cardiomyocytes to measure resting tension. Diabetic heart failure patients had higher diastolic left ventricular stiffness irrespective of LVEF. Diabetes mellitus increased the myocardial collagen volume fraction only in patients with reduced LVEF (from 14.6±1.0% to 22.4±2.2%, P<0.001) and increased cardiomyocyte resting tension only in patients with normal LVEF (from 5.1±0.7 to 8.5±0.9 kN/m2, P=0.006). Diabetes increased myocardial AGE deposition in patients with reduced LVEF (from 8.8±2.5 to 24.1±3.8 score/mm2; P=0.005) and less so in patients with normal LVEF (from 8.2±2.5 to 15.7±2.7 score/mm2, P=NS). Conclusions— Mechanisms responsible for the increased diastolic stiffness of the diabetic heart differ in heart failure with reduced and normal LVEF: Fibrosis and AGEs are more important when LVEF is reduced, whereas cardiomyocyte resting tension is more important when LVEF is normal.

Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28u2003h‐old zebrafish embryos were infected with DsRed‐labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild‐type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage‐like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non‐pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage‐like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat‐killed, wild‐type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non‐invasive and real time analysis of bacterial infections in a vertebrate host.

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.

Mycobacterial genomes contain two unique gene families, the so‐called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT‐6‐like secretion system. This cluster, designated ESX‐5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M.u2003smegmatis. Only introduction of the complete ESX‐5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX‐5 mutant of M.u2003marinum was affected in spreading to uninfected macrophages.

Enhanced number and activity of mitochondria in multiple sclerosis lesions.

Mitochondrial dysfunction has been implicated in the development and progression of multiple sclerosis (MS) lesions. Mitochondrial alterations might occur as a response to demyelination and inflammation, since demyelination leads to an increased energy demand in axons and could thereby affect the number, distribution and activity of mitochondria. We have studied the expression of mitochondrial proteins and mitochondrial enzyme activity in active demyelinating and chronic inactive MS lesions. Mitochondrial protein expression and enzyme activity in active and chronic inactive MS lesions was investigated using (immuno)histochemical and biochemical techniques. The number of mitochondria and their co‐localization with axons and astrocytes within MS lesions and adjacent normal‐appearing white matter (NAWM) was quantitatively assessed. In both active and inactive lesions we observed an increase in mitochondrial protein expression as well as a significant increase in the number of mitochondria. Mitochondrial density in axons and astrocytes was significantly enhanced in active lesions compared to adjacent NAWM, whereas a trend was observed in inactive lesions. Complex IV activity was strikingly up‐regulated in MS lesions compared to control white matter and, to a lesser extent, NAWM. Finally, we demonstrated increased immunoreactivity of the mitochondrial stress protein mtHSP70 in MS lesions, particularly in astrocytes and axons. Our data indicate the occurrence of severe mitochondrial alterations in MS lesions, which coincides with enhanced mitochondrial oxidative stress. Together, these findings support a mechanism whereby enhanced density of mitochondria in MS lesions might contribute to the formation of free radicals and subsequent tissue damage. Copyright

Lipoic Acid Affects Cellular Migration into the Central Nervous System and Stabilizes Blood-Brain Barrier Integrity

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant α-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS.

Impaired Diastolic Function After Exchange of Endogenous Troponin I with C-Terminal Truncated Troponin I in Human Cardiac Muscle

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI1–192) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 &mgr;m, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic &bgr;-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI1–192 in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca2+ sensitivity of the contractile apparatus was increased in preparations containing cTnI1–192 (pCa50: 5.73±0.03 versus 5.52±0.03 for cTnI1–192 and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI1–192-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

Reactive Oxygen Species Precede Protein Kinase C-δ Activation Independent of Adenosine Triphosphate–sensitive Mitochondrial Channel Opening in Sevoflurane-induced Cardioprotection

BackgroundIn the current study, the authors investigated the distinct role and relative order of protein kinase C (PKC)-&dgr;, adenosine triphosphate–sensitive mitochondrial K+ (mito K+ATP) channels, and reactive oxygen species (ROS) in the signal transduction of sevoflurane-induced cardioprotection and specifically addressed their mechanistic link. MethodsIsolated rat trabeculae were preconditioned with 3.8% sevoflurane and subsequently subjected to an ischemic protocol by superfusion of trabeculae with hypoxic, glucose-free buffer (40 min) followed by 60 min of reperfusion. In addition, the acute affect of sevoflurane on PKC-&dgr; and PKC-&egr; translocation and nitrotyrosine formation was established with use of immunofluorescent analysis. The inhibitors chelerythrine (6 &mgr;m), rottlerin (1 &mgr;m), 5-hydroxydecanoic acid sodium (100 &mgr;m), and n-(2-mercaptopropionyl)-glycine (300 &mgr;m) were used to study the particular role of PKC, PKC-&dgr;, mito K+ATP, and ROS in sevoflurane-related intracellular signaling. ResultsPreconditioning of trabeculae with sevoflurane preserved contractile function after ischemia. This contractile preservation was dependent on PKC-&dgr; activation, mito K+ATP channel opening, and ROS production. In addition, on acute stimulation by sevoflurane, PKC-&dgr; but not PKC-&egr; translocated to the sarcolemmal membrane. This translocation was inhibited by PKC inhibitors and ROS scavenging but not by inhibition of mito K+ATP channels. Furthermore, sevoflurane directly induced nitrosylation of sarcolemmal proteins, suggesting the formation of peroxynitrite. ConclusionsIn sevoflurane-induced cardioprotection, ROS release but not mito K+ATP channel opening precedes PKC-&dgr; activation. Sevoflurane induces sarcolemmal nitrotyrosine formation, which might be involved in the recruitment of PKC-&dgr; to the cell membrane.

Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-β-d-Ribofuranoside in the Muscle Microcirculation Increases Nitric Oxide Synthesis and Microvascular Perfusion

Objective—To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. Methods and Results—In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1-&bgr;-d-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries (≈100 &mgr;m) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19±4% at 2 mmol/L, P<0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-l-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (≈60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Conclusion—Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

BackgroundHyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes.MethodsH9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and ATP concentrations.ResultsWe found that incubation of H9c2 cells with 0.1xa0mM D,L-Hcy (=xa060xa0μM l-Hcy) resulted in an increase of ΔΨm as well as ATP concentrations. 1.1xa0mM d,l-Hcy (=xa0460xa0μM l-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73xa0mM d,l-Hcy (=xa01.18xa0mM l-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC.ConclusionWe found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.