Fons Verheyen

Hibernating Myocardium Chronically Adapted to Ischemia but Vulnerable to Sudden Death

Abstract— The inability to reproduce spontaneous ventricular fibrillation in an animal model of chronic coronary artery disease has limited advances in understanding mechanisms of sudden cardiac death (SCD). Swine with hibernating myocardium arising from a chronic left anterior descending coronary artery (LAD) occlusion have a high rate of SCD that parallels the poor clinical survival of medically treated patients with hibernating myocardium. Kaplan-Meier analysis (n = 426) demonstrated a cumulative mortality of 49% after 5 months that was almost entirely attributable to spontaneous SCD. Using implantable loop recorders, ventricular fibrillation was documented as the arrhythmic mechanism of death in all animals (n = 10) and was usually preceded by ventricular tachycardia (n = 8). Physiological studies before SCD (n = 7) demonstrated total LAD occlusion and collateral-dependent myocardium (n = 5), excluding acute occlusion as a major trigger of arrhythmia. The physiological substrate of hibernating myocardium was present before SCD, with reductions in LAD perfusion (SCD 0.79±0.13 versus 0.80±0.08 mL/min per g) and wall thickening (SCD 28±3% versus 22±3%) that were similar to survivors (n = 14). Triphenyltetrazolium chloride infarcts among animals with SCD were infrequent (4 of 32) and small, averaging 4.6% of LV mass. Histology (n = 4) showed postmortem changes but no acute inflammation nor contraction band necrosis. These data support the notion that hibernating myocardium is a pathophysiological substrate at high risk of SCD. This is independent of changes in functional stenosis severity, acute myocardial necrosis, or fibrotic scar. Thus, regional adaptations that promote myocyte survival in the setting of chronic repetitive ischemia result in a substrate with enhanced vulnerability to lethal arrhythmias and SCD.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

Proteasome Inhibition with Bortezomib Depletes Plasma Cells and Autoantibodies in Experimental Autoimmune Myasthenia Gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

Distinct contractile and molecular differences between two goat models of atrial dysfunction: AV block-induced atrial dilatation and atrial fibrillation

Atrial dilatation is an independent risk factor for thromboembolism in patients with and without atrial fibrillation (AF). In many patients, atrial dilatation goes along with depressed contractile function of the dilated atria. While some mechanisms causing atrial contractile dysfunction in fibrillating atria have been addressed previously, the cellular and molecular mechanisms of atrial contractile remodeling in dilated atria are unknown. This study characterized in vivo atrial contractile function in a goat model of atrial dilatation and compared it to a goat model of AF. Differences in the underlying mechanisms were elucidated by studying contractile function, electrophysiology and sarcoplasmic reticulum (SR) Ca2+ load in atrial muscle bundles and by analyzing expression and phosphorylation levels of key Ca2+-handling proteins, myofilaments and the expression and activity of their upstream regulators. In 7 chronically instrumented, awake goats atrial contractile dysfunction was monitored during 3 weeks of progressive atrial dilatation after AV-node ablation (AV block goats (AVB)). In open chest experiments atrial work index (AWI) and refractoriness were measured (10 goats with AVB, 5 goats with ten days of AF induced by repetitive atrial burst pacing (AF), 10 controls). Isometric force of contraction (FC), transmembrane action potentials (APs) and rapid cooling contractures (RCC, a measure of SR Ca2+ load) were studied in right atrial muscle bundles. Total and phosphorylated Ca2+-handling and myofilament protein levels were quantified by Western blot. In AVB goats, atrial size increased by 18% (from 26.6+/-4.4 to 31.6+/-5.5 mm, n=7 p<0.01) while atrial fractional shortening (AFS) decreased (from 18.4+/-1.7 to 12.8+/-4.0% at 400 ms, n=7, p<0.01). In open chest experiments, AWI was reduced in AVB and in AF goats compared to controls (at 400 ms: 8.4+/-0.9, n=7, and 3.2+/-1.8, n=5, vs 18.9+/-5.3 mmxmmHg, n=7, respectively, p<0.05 vs control). FC of isolated right atrial muscle bundles was reduced in AVB (n=8) and in AF (n=5) goats compared to controls (n=9) (at 2 Hz: 2.3+/-0.5 and 0.7+/-0.2 vs 5.5+/-1.0 mN/mm2, respectively, p<0.05). APs were shorter in AF, but unchanged in AVB goats. RCCs were reduced in AVB and AF versus control (AVB, 3.4+/-0.5 and AF, 4.1+/-1.4 vs 12.2+/-3.2 mN/mm2, p<0.05). Protein levels of protein kinase A (PKA) phosphorylated phospholamban (PLB) were reduced in AVB (n=8) and AF (n=8) vs control (n=7) by 37.9+/-12.4% and 29.7+/-10.1%, respectively (p<0.01), whereas calmodulin-dependent protein kinase II (CaMKII) phosphorylated ryanodine channels (RyR2) were increased by 166+/-55% in AVB (n=8) and by 146+/-56% in AF (n=8) goats (p<0.01). PKA-phosphorylated myosin-binding protein-C and troponin-I were reduced exclusively in AVB goat atria (by 75+/-10% and 55+/-15%, respectively, n=8, p<0.05). Atrial dilatation developing during slow ventricular rhythm after complete AV block as well as AF-induced remodeling are associated with atrial contractile dysfunction. Both AVB and AF goat atria show decreased SR Ca2+ load, likely caused by PLB dephosphorylation and RYR2 hyperphosphorylation. While shorter APs further compromise contractility in AF goat atria, reduced myofilament phosphorylation may impair contractility in AVB goat atria. Thus, atrial hypocontractility appears to have distinct molecular contributors in different types of atrial remodeling.

Perilipin 2 Improves Insulin Sensitivity in Skeletal Muscle Despite Elevated Intramuscular Lipid Levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Absence of Thrombospondin-2 Causes Age-Related Dilated Cardiomyopathy

Background— The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. Methods and Results— TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene–mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. Conclusion— TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.

Level of Activation of the Unfolded Protein Response Correlates With Paneth Cell Apoptosis in Human Small Intestine Exposed to Ischemia/Reperfusion

BACKGROUND & AIMS In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. METHODS Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. RESULTS In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. CONCLUSIONS ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.

Internalization of Modified Lipids by CD36 and SR-A Leads to Hepatic Inflammation and Lysosomal Cholesterol Storage in Kupffer Cells

Background & Aims Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs). Methods & Results Ldlr−/− mice were transplanted with wild-type (Wt), Cd36−/− or Msr1−/− bone marrow and fed a Western diet for 3months. Cd36−/−- and Msr1−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36−/−- and Msr1−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation. Conclusion CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon

Objective Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. Design In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1β and tumour necrosis factor α. Results In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. Conclusions Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

The Cholesterol Derivative 27-Hydroxycholesterol Reduces Steatohepatitis in Mice

BACKGROUND & AIMS Non-alcoholic steatohepatitis is characterized by hepatic steatosis with inflammation. Although steatosis is benign and reversible, inflammation can increase liver damage. Hepatic inflammation has been associated with accumulation of cholesterol in lysosomes of Kupffer cells. 27-Hydroxycholesterol (27HC), a derivative of cholesterol formed by CYP27A1, can mobilize cholesterol from the lysosomes to the cytoplasm. We investigated whether 27HC can change the intracellular distribution cholesterol and reduce hepatic inflammation in mice. METHODS We transplanted bone marrow from irradiated wild-type or Cyp27a1(-/-) mice to mice that do not express the low density lipoprotein receptor (Ldlr(-/-)), which are hyperlipidemic; 9 weeks later, mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 3 months. In a separate experiment, Ldlr(-/-) mice were given subcutaneous injections of 27HC and placed on regular chow or HFC diets for 3 weeks. Blood and liver tissues samples were collected and analyzed for intracellular cholesterol distribution and inflammation. RESULTS In Ldlr(-/-) mice that received bone marrow transplants from Cyp27a1(-/-) mice, lysosomes of Kupfer cells had a greater accumulation of cholesterol than those of mice that received bone marrow from wild-type mice, after the HFC diet. Liver histology and gene expression analyses showed increased inflammation and liver damage in mice given bone marrow transplants from Cyp27a1(-/-) mice and placed on the HFC diet. Administration of 27HC to Ldlr(-/-) mice, following the HFC diet, reduced the accumulation of lysosomal cholesterol and hepatic inflammation, compared with mice that were not given 27HC. CONCLUSIONS Accumulation of cholesterol in lysosomes of Kupfer cells promotes hepatic inflammation in mice. The cholesterol derivative 27HC reduces accumulation of cholesterol in lysosomes and might be used to treat non-alcoholic steatohepatitis.