Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits KATP channels through CaMKII activation.

BACKGROUND: Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs. METHODS: Cultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 µg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, and the expression of phosphorylated CaMKIIδ and NOX2/gp91phox. The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique. RESULTS: In NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP. CONCLUSIONS: L5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease.

Use of decellularized porcine liver for engineering humanized liver organ.

BACKGROUND: New bioartificial liver devices are needed to supplement the limited supply of organ donors available for patients with end-stage liver disease. Here, we report the results of a pilot study aimed at developing a humanized porcine liver by transplanting second trimester human fetal hepatocytes (Hfh) co-cultured with fetal stellate cells (Hfsc) into the decellularized matrix of a porcine liver. MATERIAL AND METHODS: Ischemic livers were removed from 19 Yorkshire swine. Liver decellularization was achieved by an anionic detergent (SDS). The decellularized matrix of three separate porcine liver matrices was seeded with 3.5 × 10(8) and 1 × 10(9) of Hfsc and Hfh, respectively, and perfused for 3, 7, and 13 d. The metabolic and synthetic activities of the engrafted cells were assessed during and after perfusion. RESULTS: Immunohistologic examination of the decellularized matrix showed removal of nuclear materials with intact architecture and preserved extracellular matrix (ECM) proteins. During perfusion of the recellularized matrices, measurement of metabolic parameters (i.e., oxygen concentration, glucose consumption, and lactate and urea production) indicated active metabolism. The average human albumin concentration was 29.48 ± 7.4 µg/mL. Immunohistochemical analysis revealed cell differentiation into mature hepatocytes. Moreover, 40% of the engrafted cells were actively proliferating, and less than 30% of cells were apoptotic. CONCLUSION: We showed that our decellularization protocol successfully removed the cellular components of porcine livers while preserving the native architecture and most ECM protein. We also demonstrated the ability of the decellularized matrix to support and induce phenotypic maturation of engrafted Hfh in a continuously perfused system.

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor-mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine-induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada -/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy.

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

Magnetic Resonance Imaging of Atherosclerotic Plaque at Clinically Relevant Field Strengths (1T) by Targeting the Integrin α4ß1.

Inflammation drives the degradation of atherosclerotic plaque, yet there are no non-invasive techniques available for imaging overall inflammation in atherosclerotic plaques, especially in the coronary arteries. To address this, we have developed a clinically relevant system to image overall inflammatory cell burden in plaque. Here, we describe a targeted contrast agent (THI0567-targeted liposomal-Gd) that is suitable for magnetic resonance (MR) imaging and binds with high affinity and selectivity to the integrin α4ß1(very late antigen-4, VLA-4), a key integrin involved in recruiting inflammatory cells to atherosclerotic plaques. This liposomal contrast agent has a high T1 relaxivity (~2 × 105 mM-1s-1 on a particle basis) resulting in the ability to image liposomes at a clinically relevant MR field strength. We were able to visualize atherosclerotic plaques in various regions of the aorta in atherosclerosis-prone ApoE-/- mice on a 1 Tesla small animal MRI scanner. These enhanced signals corresponded to the accumulation of monocyte/macrophages in the subendothelial layer of atherosclerotic plaques in vivo, whereas non-targeted liposomal nanoparticles did not demonstrate comparable signal enhancement. An inflammatory cell-targeted method that has the specificity and sensitivity to measure the inflammatory burden of a plaque could be used to noninvasively identify patients at risk of an acute ischemic event.

Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart.

We investigated whether decreased responsiveness of the heart to physiological increases in fatty acid availability results in lipid accumulation and lipotoxic heart disease. Lean and obese Zucker rats were either fed ad libitum or fasted overnight. Fasting increased plasma nonesterified fatty acid levels in both lean and obese rats, although levels were greatest in obese rats regardless of nutritional status. Despite increased fatty acid availability, the mRNA transcript levels of peroxisome proliferator-activated receptor (PPAR)-alpha-regulated genes were similar in fed lean and fed obese rat hearts. Fasting increased expression of all PPAR-alpha -regulated genes in lean Zucker rat hearts, whereas, in obese Zucker rat hearts, muscle carnitine palmitoyltransferase and medium-chain acyl-CoA dehydrogenase were unaltered with fasting. Rates of oleate oxidation were similar for hearts from fed rats. However, fasting increased rates of oleate oxidation only in hearts from lean rats. Dramatic lipid deposition occurred within cardiomyocytes of obese, but not lean, Zucker rats upon fasting. Cardiac output was significantly depressed in hearts isolated from obese rats compared with lean rats, regardless of nutritional status. Fasting increased cardiac output in hearts of lean rats only. Thus, the heart's inability to increase fatty acid oxidation in proportion to increased fatty acid availability is associated with lipid accumulation and contractile dysfunction of the obese Zucker rat.

Neuronally released vasoactive intestinal polypeptide alters atrial electrophysiological properties and may promote atrial fibrillation.

BACKGROUND: Vagal hyperactivity promotes atrial fibrillation (AF), which has been almost exclusively attributed to acetylcholine. Vasoactive intestinal polypeptide (VIP) and acetylcholine are neurotransmitters co-released during vagal stimulation. Exogenous VIP has been shown to promote AF by shortening action potential duration (APD), increasing APD spatial heterogeneity, and causing intra-atrial conduction block. OBJECTIVE: The purpose of this study was to investigate the effects of neuronally released VIP on atrial electrophysiologic properties during vagal stimulation. METHODS: We used a specific VIP antagonist (H9935) to uncover the effects of endogenous VIP released during vagal stimulation in canine hearts. RESULTS: H9935 significantly attenuated (1) the vagally induced shortening of atrial effective refractory period and widening of atrial vulnerability window during stimulation of cervical vagosympathetic trunks (VCNS) and (2) vagal effects on APD during stimulation through fat-pad ganglion plexus (VGPS). Atropine completely abolished these vagal effects during VCNS and VGPS. In contrast, VGPS-induced slowing of local conduction velocity was completely abolished by either VIP antagonist or atropine. In pacing-induced AF during VGPS, maximal dominant frequencies and their spatial gradients were reduced significantly by H9935 and, more pronouncedly, by atropine. Furthermore, VIP release in the atria during vagal stimulation was inhibited by atropine, which may account for the concealment of VIP effects with muscarinic blockade. CONCLUSION: Neuronally released VIP contributes to vagal effects on atrial electrophysiologic properties and affects the pathophysiology of vagally induced AF. Neuronal release of VIP in the atria is inhibited by muscarinic blockade, a novel mechanism by which VIP effects are concealed by atropine during vagal stimulation.

Ionic mechanisms underlying the effects of vasoactive intestinal polypeptide on canine atrial myocardium.

BACKGROUND: Vasoactive intestinal polypeptide (VIP) is released from intracardiac neurons during vagal stimulation, ischemia, and heart failure, which are associated with increased vulnerability to atrial fibrillation. VIP shortens atrial effective refractory periods in dogs. Endogenous VIP contributes to vagally mediated acceleration of atrial electric remodeling. VIP is also shown to prolong the duration of acetylcholine-induced atrial fibrillation. However, the ionic mechanisms underlying VIP effects are largely unknown. METHODS AND RESULTS: The effects of VIP on transmembrane ion channels were studied in canine atrial cardiomyocytes using patch-clamp techniques. VIP increased delayed rectifier K+ current and L-type calcium current but decreased the transient outward K+ current and sodium current. Optical mapping technique was used to assess effects of VIP on action potential durations (APDs) in isolated canine left atria. VIP shortened APD and slowed conduction velocity in a dose-dependent manner. Furthermore, VIP increased spatial heterogeneity of APD and conduction velocity, as assessed by the SDs of APD and conduction velocity, and atrial fibrillation inducibility. CONCLUSIONS: Through its diverse effects on ion channels, VIP shortens APD with increased APD spatial heterogeneity and decreases intra-atrial conduction velocity, which may play an important role in the pathogenesis of atrial arrhythmias in scenarios where VIP release is increased.

Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice.

BACKGROUND: Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. PRINCIPAL FINDINGS: By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. SIGNIFICANCE: In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.

Autoantibody from women with preeclampsia induces soluble Fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/nuclear factor of activated T-cells signaling.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT(1)) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT(1) receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT(1) receptor to induce sFlt-1 synthesis and secretion by AT(1)-receptor activating autoantibodies. AT(1)-receptor activating autoantibody-induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

alpha-1-syntrophin mutation and the long-QT syndrome: a disease of sodium channel disruption.

BACKGROUND: Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-alpha(1) (SNTA1) is known to interact with the cardiac sodium channel (hNa(v)1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNa(v)1.5 by secondarily disturbing sodium channel function. METHODS AND RESULTS: Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc> or =480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G-SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNa(v)1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation (V(h)) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1. CONCLUSION: SNTA1 is a new susceptibility gene for LQTS. A257G-SNTA1 can cause gain-of-function of Na(v)1.5 similar to the LQT3.

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1.

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.

The essential role of MEKK3 signaling in angiotensin II-induced calcineurin/nuclear factor of activated T-cells activation.

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.

Hypoxia-induced switches of myosin heavy chain iso-gene expression in rat heart.

Cardiac hypertrophy and atrophy increase expression of fetal iso-genes. A common factor is a decrease in cellular oxygen tension. To test the hypothesis that hypoxia changes cardiac MHC iso-gene expression Wistar rats were exposed to 24 and 48 h of hypobaric hypoxia (11% oxygen) and mRNA was isolated from the left ventricle. In addition, neonatal rat cardiomyocytes were incubated for up to 48 h in a hypoxic chamber. Transcript levels of MHCalpha (adult isoform), MHCbeta (fetal isoform), and Nkx2.5, the earliest known marker for cardiogenesis, were measured by real-time quantitative RT-PCR and normalized to levels of 18S rRNA. Expression of the transcription factor Nkx2.5 increased with hypoxia. Hypoxia decreased MHCalpha and increased MHCbeta transcript levels, both in vivo and in vitro. We conclude that hypoxia per se induces a pattern of isoform gene expression associated with early cardiac development.

Cardiomyocyte-specific desmin rescue of desmin null cardiomyopathy excludes vascular involvement.

Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.