Monoamine Oxidase A Contributes to Serotonin-But Not Norepinephrine-Dependent Damage of Rat Ventricular Myocytes.

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates such as catecholamines. Our study evaluates the role of MAO-A in serotonin- and norepinephrine-dependent cell damage. Experiments were performed in vivo to study the regulation of MAOA and MAOB expression and in vitro on isolated cultured adult rat ventricular cardiomyocytes (cultured for 24 h) to study the function of MAO-A. MAOA but not MAOB expression increased in maladaptive hypertrophic stages. Serotonin and norepinephrine induced morphologic cell damage (loss of rod-shaped cell structure). However, MAO-A inhibition suppressed serotonin-dependent but not norepinephrine-dependent damages. Serotonin but not norepinephrine caused a reduction in cell shortening in nondamaged cells. Serotonin induced mitochondria-dependent oxidative stress. In vivo, MAOA was induced during aging and hypertension but the expression of the corresponding serotonin uptake receptor (SLC6A4) was reduced and enzymes that reduce either oxidative stress (CAT) or accumulation of 5-hydroxyindolacetaldehyde (ALDH2) were induced. In summary, the data show that MAO-A potentially affects cardiomyocytes' function but that serotonin is not necessarily the native substrate.

Importance of Mitochondria in Cardiac Pathologies: Focus on Uncoupling Proteins and Monoamine Oxidases.

On the one hand, reactive oxygen species (ROS) are involved in the onset and progression of a wide array of diseases. On the other hand, these are a part of signaling pathways related to cell metabolism, growth and survival. While ROS are produced at various cellular sites, in cardiomyocytes the largest amount of ROS is generated by mitochondria. Apart from the electron transport chain and various other proteins, uncoupling protein (UCP) and monoamine oxidases (MAO) have been proposed to modify mitochondrial ROS formation. Here, we review the recent information on UCP and MAO in cardiac injuries induced by ischemia-reperfusion (I/R) as well as protection from I/R and heart failure secondary to I/R injury or pressure overload. The current data in the literature suggest that I/R will preferentially upregulate UCP2 in cardiac tissue but not UCP3. Studies addressing the consequences of such induction are currently inconclusive because the precise function of UCP2 in cardiac tissue is not well understood, and tissue- and species-specific aspects complicate the situation. In general, UCP2 may reduce oxidative stress by mild uncoupling and both UCP2 and UCP3 affect substrate utilization in cardiac tissue, thereby modifying post-ischemic remodeling. MAOs are important for the physiological regulation of substrate concentrations. Upon increased expression and or activity of MAOs, however, the increased production of ROS and reactive aldehydes contribute to cardiac alterations such as hypertrophy, inflammation, irreversible cardiomyocyte injury, and failure.

Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1ß release by mononuclear phagocytes.

Objective: The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in host defense against infections. High systemic IL-1ß levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1ß release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1ß release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Methods: Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1ß was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. Results: The inhibitory effect of nAChR agonists on the BzATP-induced IL-1ß release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1ß release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1ß release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. Conclusion: We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1ß release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

Effect of Metabolic Adaptation by Voluntary Running Wheel Activity and Aldosterone Inhibition on Renal Function in Female Spontaneously Hypertensive Rats.

Metabolic effects of physical activity may be reno-protective in the context of hypertension, although exercise stresses kidneys. Aldosterone participates in renal disease in hypertension, but exercise affects the plasma concentration of aldosterone. This study was designed to evaluate whether physical activity and pharmacological treatment by aldosterone have additive effects on renal protection in hypertensive rats. Female spontaneously hypertensive rats (SHR) or normotensive Wistar rats performed voluntary running wheel activity alone or in combination with aldosterone blockade (spironolactone). The following groups were studied: young and pre-hypertensive SHR (n = 5 sedentary; n = 10 running wheels, mean body weight 129 g), 10-month-old Wistar rats (n = 6 sedentary; n = 6 running wheels, mean body weight 263 g), 10-month-old SHRs (n = 18 sedentary, mean body weight 224 g; n = 6 running wheels, mean body weight 272 g; n = 6 aldosterone, mean body weight 219 g; n = 6 aldosterone and running wheels, mean body weight 265 g). Another group of SHRs had free access to running wheels for 6 months and kept sedentary for the last 3 months (n = 6, mean body weight 240 g). Aldosterone was given for the last 4 months. SHRs from the running groups had free access to running wheels beginning at the age of 6 weeks. Renal function was analyzed by microalbuminuria (Alb/Cre), urinary secretion of kidney injury molecule-1 (uKim-1), and plasma blood urea nitrogen (BUN) concentration. Molecular adaptation of the kidney to hypertension and its modification by spironolactone and/or exercise were analyzed by real-time PCR, immunoblots, and histology. After six months of hypertension, rats had increased Alb/Cre and BUN but normal uKim-1. Voluntary free running activity normalized BUN but not Alb/Cre, whereas spironolactone reduced Alb/Cre but not BUN. Exercise constitutively increased renal expression of proprotein convertase subtilisin/kexin type 9 (PCSK9; mRNA and protein) and arginase-2 (mRNA). Spironolactone reduced these effects. uKim-1 increased in rats performing voluntary running wheel activity exercise irrespectively of blood pressure and aldosterone blockade. We observed independent but no additive effects of aldosterone blockade and physical activity on renal function and on molecules potentially affecting renal lipid metabolism.

Multivalent stimulation of ß1-, but not ß2-receptors by adrenaline functionalised gold nanoparticles.

In this study, we present a strategy for the synthesis of catecholamine functionalised gold nanoparticles and investigated their multivalent interactions with adrenergic receptors in different biological systems. The catecholamines adrenaline and noradrenaline represent key examples of adrenergic agonists. We used gold nanoparticles as carriers and functionalised them on their surface with a variety of these neurotransmitter molecules. For this purpose, we synthesised each ligand separately using mercaptoundecanoic acid as a bifunctional linking unit and adrenaline (or noradrenaline) as a biogenic amine. This ligand was then immobilised onto the surface of presynthesised spherical monodispersive gold nanoparticles in a ligand exchange reaction. After detailed analytical characterisations, the functionalised gold nanoparticles were investigated for their interactions with adrenergic receptors in intestinal, cardiac and respiratory tissues. Whereas the contractility of respiratory smooth muscle cells (regulated by ß2-receptors) was not influenced, (nor)adrenaline functionalised nanoparticles administered in nanomolar concentrations induced epithelial K+ secretion (mediated via different ß-receptors) and increased contractility of isolated rat cardiomyocytes (mediated by ß1-receptors). The present results suggest differences in the accessibility of adrenergic agonists bound to gold nanoparticles to the binding pockets of different ß-receptor subtypes.

Effect of Anti-Hypertensive Medication on Plasma Concentrations of Lysyl Oxidase: Evidence for Aldosterone-IL-6-Dependent Regulation of Lysyl Oxidase Blood Concentration.

Lysyl oxidase (LOX) is a secretory protein that catalyzes elastin and collagen cross-linking. Lowering LOX expression and activity in endothelial cells is associated with a high risk of aneurysms and vascular malformation. Interleukin-6 (IL-6), elevated in hypertension, is known to suppress LOX expression. The influence of anti-hypertensive medication on the plasma LOX concentration is currently unknown. In a cohort of 34 patients diagnosed with resistant hypertension and treated with up to nine different drugs, blood concentration of LOX was analyzed to identify drugs that have an impact on plasma LOX concentration. Key findings were confirmed in a second independent patient cohort of 37 patients diagnosed with dilated cardiomyopathy. Blood concentrations of aldosterone and IL-6 were analyzed. In vitro, the effect of IL-6 on LOX expression was analyzed in endothelial cells. Patients receiving aldosterone antagonists had the highest plasma LOX concentration in both cohorts. This effect was independent of sex, age, blood pressure, body mass index, and co-medication. Blood aldosterone concentration correlates with plasma IL-6 concentration. In vitro, IL-6 decreased the expression of LOX in endothelial cells but not fibroblasts. Aldosterone was identified as a factor that affects blood concentration of LOX in an IL-6-dependent manner.

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo.

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

Comparison of the Anti-inflammatory Properties of Two Nicotinic Acetylcholine Receptor Ligands, Phosphocholine and pCF3-diEPP.

Activation of nicotinic acetylcholine receptors (nAChRs) expressed by innate immune cells can attenuate pro-inflammatory responses. Silent nAChR agonists, which down-modulate inflammation but have little or no ionotropic activity, are of outstanding clinical interest for the prevention and therapy of numerous inflammatory diseases. Here, we compare two silent nAChR agonists, phosphocholine, which is known to interact with nAChR subunits α7, α9, and α10, and pCF3-N,N-diethyl-N'-phenyl-piperazine (pCF3-diEPP), a previously identified α7 nAChR silent agonist, regarding their anti-inflammatory properties and their effects on ionotropic nAChR functions. The lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 by primary murine macrophages was inhibited by pCF3-diEPP, while phosphocholine was ineffective presumably because of instability. In human whole blood cultures pCF3-diEPP inhibited the LPS-induced secretion of IL-6, TNF-α and IL-1ß. The ATP-mediated release of IL-1ß by LPS-primed human peripheral blood mononuclear leukocytes, monocytic THP-1 cells and THP-1-derived M1-like macrophages was reduced by both phosphocholine and femtomolar concentrations of pCF3-diEPP. These effects were sensitive to mecamylamine and to conopeptides RgIA4 and [V11L; V16D]ArIB, suggesting the involvement of nAChR subunits α7, α9 and/or α10. In two-electrode voltage-clamp measurements pCF3-diEPP functioned as a partial agonist and a strong desensitizer of classical human α9 and α9α10 nAChRs. Interestingly, pCF3-diEPP was more effective as an ionotropic agonist at these nAChRs than at α7 nAChR. In conclusion, phosphocholine and pCF3-diEPP are potent agonists at unconventional nAChRs expressed by monocytic and macrophage-like cells. pCF3-diEPP inhibits the LPS-induced release of pro-inflammatory cytokines, while phosphocholine is ineffective. However, both agonists signal via nAChR subunits α7, α9 and/or α10 to efficiently down-modulate the ATP-induced release of IL-1ß. Compared to phosphocholine, pCF3-diEPP is expected to have better pharmacological properties. Thus, low concentrations of pCF3-diEPP may be a therapeutic option for the treatment of inflammatory diseases including trauma-induced sterile inflammation.

Oral application of levosimendan before ischaemia/reperfusion with or without cardioplegic arrest in rats.

OBJECTIVES: Clinical studies have indicated minor beneficial effects of the calcium sensitizer levosimendan on clinical outcomes in patients undergoing cardiac surgery. Here, the influence of levosimendan administered 24 h before cardiac arrest on myocardial function was examined in rat hearts perfused in a Langendorff model. METHODS: Levosimendan (Levo group) or NaCl (control group) was administered to 53 rats via drinking water 24 h prior to mounting excised hearts on a Langendorff apparatus. Cardiac arrest with or without cardioplegia was induced in both groups; another set of hearts was perfused continuously. During 90-min reperfusion at 36°C, functional parameters were measured and normalized to baseline values. Troponin I was quantified in coronary sinus effluent, and the functionality of isolated cardiomyocytes was studied. RESULTS: Oral application of levosimendan showed therapeutic efficacy. Baseline values were similar in the Levo and NaCl groups except for coronary flow. After ischaemia and reperfusion, Levo hearts did not recover better than NaCl hearts {left ventricular derived pressure: 63 [standard deviation (SD): 36.2] vs 46 (SD: 41.8)% baseline; P = 0.386}, In hearts exposed to cardioplegia, functional recovery only slightly differed in the Levo and NaCl groups [left ventricular derived pressure: 69.96 (SD: 12.7) vs 51.89 (SD: 28.1)% baseline; P = 0.09]. Cell shortening of cardiomyocytes isolated from hearts exposed to ischaemia or perfusion was better in Levo groups [cell shortening: 7.65 (SD: 1.95) %; 7.8 (SD: 1.79)% vs 6.28 (SD: 1.67)%; 6.5 (SD: 1.87)%, P < 0.001]; this benefit was absent in cardioplegia-treated hearts. CONCLUSIONS: Levosimendan applied orally before ischaemia/reperfusion improves functional recovery, but this effect is only moderate when cardioplegia is included. Differences between hearts exposed to cardioplegia or to global ischaemia may indicate why levosimendan-related beneficial effects do not directly translate into better clinical outcome.

Uncoupling Proteins in Striated Muscle Tissue: Known Facts and Open Questions.

Significance: Uncoupling proteins (UCPs) are a family of proteins that allow proton leakage across the inner mitochondrial membrane. Although UCP1, also known as thermogenin, is well known and important for heat generation in brown adipose tissue, striated muscles express two distinct members of UCP, namely UCP2 and UCP3. Unlike UCP1, the main function of UCP2 and UCP3 does not appear to be heat production. Recent Advances: Interestingly, UCP2 is the main isoform expressed in cardiac tissues, whereas UCP3 is the dominant isoform in skeletal muscles. In the past years, researchers have started to investigate the regulation of UCP2 and UCP3 expression in striated muscles. Furthermore, concepts about the proposed functions of UCP2 and UCP3 in striated muscles are developed but are still a matter of debate. Critical Issues: Potential functions of UCP2 and UCP3 in striated muscles include a role in protection against mitochondria-dependent oxidative stress, as transporter for pyruvate, fatty acids, and protons into and out of the mitochondria, and in metabolic sensing. In this context, the different isoform expression of UCP2 and UCP3 in the skeletal and cardiac muscle may be related to different metabolic requirements of the two organs. Future Directions: The level of expression of UCP2 and UCP3 in striated muscles changes in different disease stages. This suggests that UCPs may become drug targets for therapy in the future. Antioxid. Redox Signal. 37, 324-335.

Effects of voluntary exercise on the expression of browning markers in visceral and subcutaneous fat tissue of normotensive and spontaneously hypertensive rats.

High physical activity is important to optimize the function of adipose tissue. Dysfunctional adipose tissue contributes to the development of metabolic stress, chronic inflammation, and hypertension. To improve our current understanding of the interaction between physical exercise and adipose tissue, we analyzed the effect of 10 months voluntary running wheel activity of rats on uncoupling protein (UCP) 1 negative white adipose tissue (visceral and subcutaneous adipose tissue, VWAT and SWAT). Analysis was performed via RT-PCR and immunoblot from adipose tissues depicted from adult normotensive and spontaneously hypertensive female rats. UCP1 negative VWAT differed from UCP1 positive WAT and brown adipose tissue (BAT) from interscapular fat depots, by lacking the expression of UCP1 and low expression of Cidea, a transcriptional co-activator of UCP1. High physical activity affected the expression of five genes in SWAT (Visfatin (up), RBP5, adiponectin, Cidea, and Nrg4 (all down)) but only one gene (Visfatin, up) in VWAT. Furthermore, the expression of these genes is differentially regulated in VWAT and SWAT of normotensive and spontaneously hypertensive rats (SHR) under sedentary conditions (UCP2) and exercise (Visfatin, Cidea, Nrg4). Keeping the animals after 6 months of voluntary exercise under observation for an additional period of 4 months without running wheels, Visfatin, Cidea, and Nrg4 were stronger expressed in VWAT of SHRs than in sedentary control rats. In summary, our study shows that SWAT is more responsible to exercise than VWAT.

Uric Acid Deteriorates Load-Free Cell Shortening of Cultured Adult Rat Ventricular Cardiomyocytes via Stimulation of Arginine Turnover.

Hyperuricemia is a risk factor for heart disease. Cardiomyocytes produce uric acid via xanthine oxidase. The enzymatic reaction leads to oxidative stress in uric-acid-producing cells. However, extracellular uric acid is the largest scavenger of reactive oxygen species, specifically to nitrosative stress, which can directly affect cells. Here, the effect of plasma-relevant concentrations of uric acid on adult rat ventricular cardiomyocytes is analyzed. A concentration- and time-dependent reduction of load-free cell shortening is found. This is accompanied by an increased protein expression of ornithine decarboxylase, the rate-limiting enzyme of the polyamine metabolism, suggesting a higher arginine turnover. Subsequently, the effect of uric acid was attenuated if other arginine consumers, such as nitric oxide synthase, are blocked or arginine is added. In the presence of uric acid, calcium transients are increased in cardiomyocytes irrespective of the reduced cell shortening, indicating calcium desensitization. Supplementation of extracellular calcium or stimulation of intracellular calcium release by ß-adrenergic receptor stimulation attenuates the uric-acid-dependent effect. The effects of uric acid are attenuated in the presence of a protein kinase C inhibitor, suggesting that the PKC-dependent phosphorylation of troponin triggers the desensitizing effect. In conclusion, high levels of uric acid stress cardiomyocytes by accelerating the arginine metabolism via the upregulation of ornithine decarboxylase.

Mitochondria Isolated from Hearts Subjected to Ischemia/Reperfusion Benefit from Adenine Nucleotide Translocase 1 Overexpression.

Reperfusion is the only feasible therapy following myocardial infarction, but reperfusion has been shown to damage mitochondrial function and disrupt energy production in the heart. Adenine nucleotide translocase 1 (ANT1) facilitates the transfer of ADP/ATP across the inner mitochondrial membrane; therefore, we tested whether ANT1 exerts protective effects on mitochondrial function during ischemia/reperfusion (I/R). The hearts of wild-type (WT) and transgenic ANT1-overexpressing (ANT1-TG) rats were exposed to I/R injury using the standard Langendorff technique, after which mitochondrial function, hemodynamic parameters, infarct size, and components of the contractile apparatus were determined. ANT1-TG hearts expressed higher ANT protein levels, with reduced levels of oxidative 4-hydroxynonenal ANT modifications following I/R. ANT1-TG mitochondria isolated from I/R hearts displayed stable calcium retention capacity (CRC) and improved membrane potential stability compared with WT mitochondria. Mitochondria isolated from ANT1-TG hearts experienced less restricted oxygen consumption than WT mitochondria after I/R. Left ventricular diastolic pressure (Pdia) decreased in ANT1-TG hearts compared with WT hearts following I/R. Preserved diastolic function was accompanied by a decrease in the phospho-lamban (PLB)/sarcoplasmic reticulum calcium ATPase (SERCA2a) ratio in ANT1-TG hearts compared with that in WT hearts. In addition, the phosphorylated (P)-PLB/PLB ratio increased in ANT1-TG hearts after I/R but not in WT hearts, which indicated more effective calcium uptake into the sarcoplasmic reticulum in ANT1-TG hearts. In conclusion, ANT1-TG rat hearts coped more efficiently with I/R than WT rat hearts, which was reflected by preserved mitochondrial energy balance, diastolic function, and calcium dynamics after reperfusion.

PI3K as Mediator of Apoptosis and Contractile Dysfunction in TGFß1-Stimulated Cardiomyocytes.

BACKGROUND: TGFß1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFß1 mediates its effect via the TGFß receptor I (ALK5) and the activation of SMAD transcription factors, but TGFß1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFß1-induced cardiomyocytes apoptosis and contractile dysfunction. METHODS AND RESULTS: Incubation of isolated ventricular cardiomyocytes with TGFß1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFß1-stimulated cells. Additionally, TGFß-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFß effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFß target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. CONCLUSIONS: TGFß1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.

Pro-inflammatory Vascular Stress in Spontaneously Hypertensive Rats Associated With High Physical Activity Cannot Be Attenuated by Aldosterone Blockade.

The effect of high physical activity, performed as voluntary running wheel exercise, on inflammation and vascular adaptation may differ between normotensive and spontaneously hypertensive rats (SHRs). We investigated the effects of running wheel activity on leukocyte mobilization, neutrophil migration into the vascular wall (aorta), and transcriptional adaptation of the vascular wall and compared and combined the effects of high physical activity with that of pharmacological treatment (aldosterone antagonist spironolactone). At the start of the 6th week of life, before hypertension became established in SHRs, rats were provided with a running wheel over a period of 10-months'. To investigate to what extent training-induced changes may underlie a possible regression, controls were also generated by removal of the running wheel for the last 4 months. Aldosterone blockade was achieved upon oral administration of Spironolactone in the corresponding treatment groups for the last 4 months. The number of circulating blood cells was quantified by FACS analysis of peripheral blood. mRNA expression of selected proteins was quantified by RT-PCR. Histology and confocal laser microscopy were used to monitor cell migration. Although voluntary running wheel exercise reduced the number of circulating neutrophils in normotensive rats, it rather increased it in SHRs. Furthermore, running wheel activity in SHRs but not normotensive rats increased the number of natural killer (NK)-cells. Except of the increased expression of plasminogen activator inhibitor (PAI)-1 and reduction of von Willebrand factor (vWF), running wheel activity exerted a different transcriptional response in the vascular tissue of normotensive and hypertensive rats, i.e., lack of reduction of the pro-inflammatory IL-6 in vessels from hypertensive rats. Spironolactone reduced the number of neutrophils; however, in co-presence with high physical activity this effect was blunted. In conclusion, although high physical activity has beneficial effects in normotensive rats, this does not predict similar beneficial effects in the concomitant presence of hypertension and care has to be taken on interactions between pharmacological approaches and high physical activity in hypertensives.

Untypical Metabolic Adaptations in Spontaneously Hypertensive Rats to Free Running Wheel Activity Includes Uncoupling Protein-3 (UCP-3) and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Expression.

Obesity and hypertension are common risk factors for cardiovascular disease whereas an active lifestyle is considered as protective. However, the interaction between high physical activity and hypertension is less clear. Therefore, this study investigates the impact of high physical activity on the muscular and hepatic expression of glucose transporters (Glut), uncoupling proteins (UCPs), and proprotein convertase subtilisin/kexin type 9 (PCSK9) in spontaneously hypertensive rats (SHRs). Twenty-four female rats (12 normotensive rats and 12 SHRs) were divided into a sedentary control and an exercising group that had free access to running wheels at night for 10 months. Blood samples were taken and blood pressure was determined. The amount of visceral fat was semi-quantitatively analyzed and Musculus gastrocnemius, Musculus soleus, and the liver were excised. Acute effects of free running wheel activity were analyzed in 15 female SHRs that were sacrificed after 2 days of free running wheel activity. M. gastrocnemius and M. soleus differed in their mRNA expression of UCP-2, UCP-3, GLUT-4, and PCSK9. Hypertension was associated with lower levels of UCP-2 and PCSK9 mRNA in the M. gastrocnemius, but increased expression of GLUT-1 and GLUT-4 in the M. soleus. Exercise down-regulated UCP-3 in the M. soleus in both strains, in the M. gastrocnemius only in normotensives. In SHRs exercise downregulated the expression of UCP-2 in the M. soleus. Exercise increased the expression of GLUT-1 in the M. gastrocnemius in both strains, and that of GLUT-4 protein in the M. soleus, whereas it increased the muscle-specific expression of PCSK9 only in normotensive rats. Effects of exercise on the hepatic expression of cholesterol transporters were seen only in SHRs. As an acute response to exercise increased expressions of the myokine IL-6 and that of GLUT-1 were found in the muscles. This study, based on transcriptional adaptations in striated muscles and livers, shows that rats perform long-term metabolic adaptations when kept with increased physical activity. These adaptations are at least in part required to stabilize normal protein expression as protein turnover seems to be modified by exercise. However, normotensive and hypertensive rats differed in their responsiveness. Based on these results, a direct translation from normotensive to hypertensive rats is not possible. As genetic differences between normotensive humans and patients with essential hypertension are likely to be present as well, we would expect similar differences in humans that may impact recommendations for non-pharmacological interventions.

Connexin 43 phosphorylation by casein kinase 1 is essential for the cardioprotection by ischemic preconditioning.

Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43MAPKmut, Cx43PKCmut, and Cx43CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43PKCmut and Cx43CK1mut but not in Cx43MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43MAPKmut, and Cx43PKCmut, but not in Cx43CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC's cardioprotection.

Cardiomyocytes-specific deletion of monoamine oxidase B reduces irreversible myocardial ischemia/reperfusion injury.

Monoamine oxidase B (MAO-B), a protein localized at the outer mitochondrial membrane, catalyzes the oxidative deamination of biogenic amines thereby producing reactive oxygen species (ROS). Increased ROS formation contributes to myocardial ischemia/reperfusion (I/R); however, the importance of different ROS producing enzymes for increased I/R-induced ROS formation and the subsequent I/R injury is still a matter of debate. Here we describe the first cardiomyocytes-specific MAO-B knockout mouse and test the hypothesis that lack of cardiomyocyte MAO-B protects the heart from I/R injury. A cardiac-specific and tamoxifen-inducible MAO-B knockout mouse (MAO-B KO) was generated using the Cre/lox system; Cre-negative MAO-Bfl/fl littermates served as controls (WT). Lack of MAO-B was verified by Western blot and immunohistochemistry. Cardiac function of MAO-B KO and WT was analyzed by echocardiography, quantification of mitochondrial ROS production, and measurement of myocardial infarct size (in % of ventricle) in hearts exposed to global I/R using the Langendorff technique. MAO-B protein expression was significantly down-regulated in MAO-B KO mice after two weeks of tamoxifen feeding followed by ten weeks of feeding with normal chow. ROS formation stimulated by the MAO-B-specific substrate ß-phenylethylamin (PEA; 250 µM) was significantly lower in mitochondria isolated from MAO-B KO compared to WT hearts (WT 4.5 ± 0.8 a. u.; MAO-B KO 1.2 ± 0.3 a. u.). Echocardiography revealed no significant differences in LV dimensions as well as ejection fraction (EF) between WT and MAO-B KO mice (EF: WT 67.3 ± 8.8%; MAO-B KO 67.7 ± 6.5%). After I/R, infarct size was significantly lower in MAO-B KO hearts (WT 69.3 ± 15.1%; MAO-B KO 46.8 ± 12.0%). CONCLUSION: Lack of cardiomyocytes-specific MAO-B reduces infarct size suggesting that MAO-B activity contributes to acute reperfusion injury.

Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes.

PURPOSE: Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. METHODS: Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. RESULTS: MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3ß (GSK3ß). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3ß-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. CONCLUSION: MMPs' inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.