Gerald Wölkart

Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase.

OBJECTIVE The role of nitric oxide (NO) in myocardial ischemia/reperfusion injury remains controversial as both NO donors and NO synthase (NOS) inhibitors have shown to be protective. We generated transgenic (TG) mice that overexpress endothelial NOS (eNOS) exclusively in cardiac myocytes to determine the effects of high cardiac NO levels on ischemia/reperfusion injury and cellular Ca(2+) homeostasis. Wild-type (WT) mice served as controls. METHODS Hearts were perfused in vitro and subjected to 20 min of total no-flow ischemia and 30 min of reperfusion (n=5 per group). Left ventricular function, cGMP levels and intracellular Ca(2+) transients (Ca(2+)(i)) were determined. RESULTS Left ventricular pressure was reduced (maximum, -33%) and basal cardiac cGMP was increased (twofold) in TG hearts, and the changes were reversed by NOS blockade with N(G)-nitro-L-arginine methyl ester (L-NAME). Relative to baseline, recovery of reperfusion contractile function was significantly better in hearts from TG (98%) than WT (51%) mice, and L-NAME abolished this effect. Heart rate and coronary perfusion pressure were not different between groups. Systolic and diastolic Ca(2+)(i) concentrations were similar in WT and TG hearts, but Ca(2+)(i) overload during early reperfusion tended to be less in TG hearts. Kinetic analysis of pressure curves and Ca(2+)(i) transients revealed a faster left ventricular diastolic relaxation and abbreviated aequorin light signals in TG hearts at baseline and during reperfusion. CONCLUSIONS High levels of NO/cGMP strongly protect against ischemia/reperfusion injury, the protection is largely independent of changes in Ca(2+)(i) modulation, but relates to reduced preischemic performance. Myocyte-specific NO augmentation may aid in studies of the (patho)physiological roles of cardiac-derived NO.

Tissue-toxic effects of phosphatidylcholine/deoxycholate after subcutaneous injection for fat dissolution in rats and a human volunteer.

BACKGROUND The safety of the lipodissolution procedure for the cosmetic treatment of fat is unknown. OBJECTIVES The objective was to determine the subcutaneous tissue effects of phosphatidylcholine solubilized with deoxycholate (PC/DC) in rats and a human volunteer. METHODS Rats were treated subcutaneously three times with 50, 300, or 600 μL of PC/DC formula on the abdomen in a chronic study (30 days). A human volunteer undergoing elective liposuction was similarly treated. Cell membrane lysis, cell viability, and histologic status were determined on fresh biopsies of subcutaneous fat from the injection sites. RESULTS PC/DC dose-dependently reduced membrane integrity and cell viability. Histologic alterations induced by PC/DC included fibroplasia, bandlike fibrosis in the region of the cutaneous muscle, and partial muscle loss. The highest dose caused widespread fat necrosis, fat cyst formation, and necrotic changes of the walls of small blood vessels. Histologic sections of subcutaneous tissue from the human volunteer showed dose-dependent panniculitis, fat cysts, and vessel necrosis. DC (2.5%), tested for comparison in the rat, exerted membrane and histologic effects similar to those of PC/DC. Solvent controls caused negligible alterations. CONCLUSIONS Injection lipolysis with PC/DC causes tissue fibrosis and necrosis of adipose and vascular tissues in rat and man, making the long-term safety of PC/DC for nonsurgical treatment of subcutaneous fat deposits uncertain.

Vascular Bioactivation of Nitroglycerin Is Catalyzed by Cytosolic Aldehyde Dehydrogenase-2

Rationale: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. Objective: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. Methods and Results: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. Conclusions: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.

Myocardial dysfunction and male mortality in peroxisome proliferator-activated receptor alpha knockout mice overexpressing lipoprotein lipase in muscle.

Free fatty acids (FFA) are liberated from triglyceride-rich lipoproteins by lipoprotein lipase (LPL) and are considered to be a principal energy source for the heart. The peroxisome proliferator-activated receptor alpha (PPARα) is a key regulator of FFA catabolism. To investigate its role in cardiac muscle metabolism, transgenic mice overexpressing LPL in skeletal and cardiac muscle were bred on a PPARα knockout background. Fifty-five percent of male animals lacking PPARα and overexpressing LPL died within 4 months after birth. In contrast, females of this genotype stayed alive. Deceased animals exhibited cardiopulmonary congestion but had no increase of neutral lipids in the heart. Changes in plasma glucose, FFA, lactate, and triglycerides did not clearly account for gender-specific differences in mortality; however, they indicated a critical role for PPARα during fasting. Analysis of cardiac function revealed that in isolated perfused hearts, left ventricular developed pressure (a measure of contractility) was markedly lower in PPARα knockout mice overexpressing LPL compared with controls. Glucose uptake of isolated perfused hearts was significantly higher in PPARα knockout mice with both normal or increased LPL expression. However, uptake of FFA was not different among genotypes. In contrast, fasted FFA levels were significantly lower in cardiac muscle of PPARα knockout mice with normal LPL expression (−26%) and PPARα knockout mice overexpressing LPL (−38%) compared with controls. Our results indicate a critical role for PPARα in myocardial pump function and suggest that mouse models combining different genetic effects such as PPARα knockout mice overexpressing muscle LPL may be useful to study cardiomyopathies.

Vascular dysfunction and myocardial contractility in the JCR:LA-corpulent rat

OBJECTIVE The JCR:LA-corpulent rat is a unique animal model of human vascular disease that exhibits a profound insulin resistance, vasculopathy, and cardiovascular dysfunction. We tested the hypothesis that the defects affect endothelial and smooth muscle function of the coronary microvasculature as well as cardiac contractility. Coronary, myocardial and aortic function were assessed in obese (homozygous for the cp gene, cp/cp) and lean (heterozygous or homozygous normal, +/?) littermates aged 7 and 18 weeks. METHODS Coronary endothelial relaxation was examined in isolated perfused hearts by determining the effect of bradykinin (0. 1-1000 nmol l(-1)) on coronary perfusion pressure (CPP), myocardial mechanical function was evaluated in terms of left-ventricular developed pressure (LVDevP), and aortic relaxation with the endothelium-dependent agonist, A 23187 (1-1000 nmol l(-1)). RESULTS In rats aged 7 weeks, bradykinin reduced CPP from 133+/-1 mmHg to 43+/-1 mmHg (-67%) in lean rats, but only to 64+/-3 mmHg (-52%) in corpulent rats (n=6, P<0.05). Similar differences were found in rats aged 18 weeks (n=8). Inhibition of NO synthase with N(G)-nitro-L-arginine (L-NNA; 0.2 mmol l(-1)) impaired, and tetrahydrobiopterin (0.1 mmol l(-1)), a NO synthase cofactor, restored relaxation in cp/cp rats. Spermine/NO equally reduced CPP in both groups (-58%). Mechanical function was similar in lean and corpulent rats, aortic endothelial relaxation was attenuated by approximately 30% and aortic smooth muscle function was normal (7 weeks) or improved (18 weeks) in the cp/cp genotype. CONCLUSION These results suggest that (i) there is a specific impairment of NO-mediated relaxation of the coronary resistance vessels in the JCR:LA-corpulent rat that is not associated with impaired baseline myocardial contractility, and (ii) exogenous tetrahydrobiopterin reversed the relaxation defects that are part of the vascular complications typical for the insulin resistance syndrome.

Cardiac dysfunction in adipose triglyceride lipase deficiency: treatment with a PPARα agonist.

Adipose triglyceride lipase (ATGL) has been identified as a rate‐limiting enzyme of mammalian triglyceride catabolism. Deletion of the ATGL gene in mice results in severe lipid accumulation in a variety of tissues including the heart. In the present study we investigated cardiac function in ATGL‐deficient mice and the potential therapeutic effects of the PPARα and γ agonists Wy14,643 and rosiglitazone, respectively.

TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1

Aim TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. Methods and results We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3′,4′-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. Conclusion Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli.

Vascular tolerance to nitroglycerin in ascorbate deficiency

AIMS Nitroglycerin (GTN) acts through release of a nitric oxide (NO)-related activator of soluble guanylate cyclase in vascular smooth muscle. Besides enzymatic GTN bioactivation catalysed by aldehyde dehydrogenase, non-enzymatic reaction of GTN with ascorbate also results in the formation of a bioactive product. Using an established guinea pig model of ascorbate deficiency, we investigated whether endogenous ascorbate contributes to GTN-induced vasodilation. METHODS AND RESULTS Guinea pigs were fed either standard or ascorbate-free diet for 2 or 4 weeks prior to measuring the GTN response of aortic rings and isolated hearts. The effects of ascorbate on GTN metabolism were studied with purified mitochondrial aldehyde dehydrogenase (ALDH2) and isolated mitochondria. Ascorbate deprivation led to severe scorbutic symptoms and loss of body weight, but had no (2 weeks) or only slight (4 weeks) effects on aortic relaxations to a direct NO donor. The EC(50) of GTN was increased from 0.058 +/- 0.018 to 0.46 +/- 0.066 and 5.5 +/- 0.9 microM after 2 and 4 weeks of ascorbate-free diet, respectively. Similarly, coronary vasodilation to GTN was severely impaired in ascorbate deficiency. The potency of GTN was reduced to a similar extent by ALDH inhibitors in control and ascorbate-deficient blood vessels. Up to 10 mM ascorbate had no effect on GTN metabolism catalysed by purified ALDH2 or liver mitochondria isolated from ascorbate-deficient guinea pigs. CONCLUSION Our results indicate that prolonged ascorbate deficiency causes tolerance to GTN without affecting NO/cyclic GMP-mediated vasorelaxation.

Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase-catalysed bioactivation of nitroglycerin

Background and purpose:  Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.

Subcutaneous injection of liquid and foamed polidocanol: extravasation is not responsible for skin necrosis during reticular and spider vein sclerotherapy

Background  Cutaneous necrosis is one of the most annoying complications of reticular and spider vein sclerotherapy. The precise incidence of the complication is not known, although various sources reported incidence between 0.2% and 1.2%. Among a few mechanisms proposed to explain it, extravasation of the sclerosant into the perivascular tissue has been cited as the major cause.