Louis Villeneuve

Characterization of a hyperpolarization‐activated time‐dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium

Cardiomyocytes from the pulmonary vein sleeves (PVs) are known to play an important role in atrial fibrillation. PVs have been shown to exhibit time‐dependent hyperpolarization‐induced inward currents of uncertain nature. We observed a time‐dependent K+ current upon hyperpolarization of PV and left atrial (LA) cardiomyocytes (IKH) and characterized its biophysical and pharmacological properties. The activation time constant was weakly voltage dependent, ranging from 386 ± 14 to 427 ± 37 ms between −120 and −90 mV, and the half‐activation voltage averaged −93 ± 4 mV. IKH was larger in PV than LA cells (e.g. at −120 mV: −2.8 ± 0.3 versus−1.9 ± 0.2 pA pF−1, respectively, P < 0.01). The reversal potential was ∼−84 mV with 5.4 mm[K+]o and changed by 55.7 ± 2.4 mV per decade [K+]o change. IKH was exquisitely Ba2+ sensitive, with a 50% inhibitory concentration (IC50) of 2.0 ± 0.3 μm (versus 76.0 ± 17.9 μm for instantaneous inward‐rectifier current, P < 0.01), and showed similar Cs+ sensitivity to instantaneous current. IKH was potently blocked by tertiapin‐Q, a selective Kir3‐subunit channel blocker (IC50 10.0 ± 2.1 nm), was unaffected by atropine and was significantly increased by isoproterenol (isoprenaline), carbachol and the non‐hydrolysable guanosine triphosphate analogue GTPγS. IKH activation by carbachol required GTP in the pipette and was prevented by pertussis toxin pretreatment. Tertiapin‐Q delayed repolarization in atropine‐exposed multicellular atrial preparations studied with standard microelectrodes (action potential duration pre‐ versus post‐tertiapin‐Q: 190.4 ± 4.3 versus 234.2 ± 9.9 ms, PV; 202.6 ± 2.6 versus 242.7 ± 6.2 ms, LA; 2 Hz, P < 0.05 each). Seven‐day atrial tachypacing significantly increased IKH (e.g. at −120 mV in PV: from −2.8 ± 0.3 to −4.5 ± 0.5 pA pF−1, P < 0.01). We conclude that IKH is a time‐dependent, hyperpolarization‐activated K+ current that likely involves Kir3 subunits and appears to play a significant role in atrial physiology.

Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

Much is known about β2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the β2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-Gβγ complexes initially associate in the endoplasmic reticulum, whereas Gα subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.

Changes in Connexin Expression and the Atrial Fibrillation Substrate in Congestive Heart Failure

Rationale: Although connexin changes are important for the ventricular arrhythmic substrate in congestive heart failure (CHF), connexin alterations during CHF-related atrial arrhythmogenic remodeling have received limited attention. Objective: To analyze connexin changes and their potential contribution to the atrial fibrillation (AF) substrate during the development and reversal of CHF. Methods and Results: Three groups of dogs were studied: CHF induced by 2-week ventricular tachypacing (240 bpm, n=15); CHF dogs allowed a 4-week nonpaced recovery interval after 2-week tachypacing (n=16); and nonpaced sham controls (n=19). Left ventricular (LV) end-diastolic pressure and atrial refractory periods increased with CHF and normalized on CHF recovery. CHF caused abnormalities in atrial conduction indexes and increased the duration of burst pacing-induced AF (DAF, from 22±7 seconds in control to 1100±171 seconds, P<0.001). CHF did not significantly alter overall atrial connexin (Cx)40 and Cx43 mRNA and protein expression levels, but produced Cx43 dephosphorylation, increased Cx40/Cx43 protein expression ratio and caused Cx43 redistribution toward transverse cell-boundaries. All of the connexin-alterations reversed on CHF recovery, but CHF-induced conduction abnormalities and increased DAF (884±220 seconds, P<0.001 versus control) remained. The atrial fibrous tissue content increased from 3.6±0.7% in control to 14.7±1.5% and 13.3±2.3% in CHF and CHF recovery, respectively (both P<0.01 versus control), with transversely running zones of fibrosis physically separating longitudinally directed muscle bundles. In an ionically based action potential/tissue model, fibrosis was able to account for conduction abnormalities associated with CHF and recovery. Conclusions: CHF causes atrial connexin changes, but these are not essential for CHF-related conduction disturbances and AF promotion, which are rather related primarily to fibrotic interruption of muscle bundle continuity.

Cellular Signaling Underlying Atrial Tachycardia Remodeling of L-type Calcium Current

Atrial tachycardia (AT) downregulates L-type Ca2+ current (ICaL) and causes atrial fibrillation–promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours. Cellular tachypacing (P3) mimicked effects of in vivo AT: decreased ICaL and transient outward current (Ito), unchanged ICaT, IKr, and IKs, and reduced action potential duration (APD). ICaL was unchanged in P3 at 2 and 8 hours but decreased by 55±6% at 24 hours. Tachypacing caused Ca2+i accumulation in P3 cells at 2 to 8 hours, but, by 24 hours, Ca2+i returned to baseline. Cav1.2 mRNA expression was not altered at 2 hours but decreased significantly at 8 and 24 hours (32±4% and 48±4%, respectively) and protein expression was decreased (47±8%) at 24 hours only. Suppressing Ca2+i increases during tachypacing with the ICaL blocker nimodipine or the Ca2+ chelator BAPTA-AM prevented ICaL downregulation. Calcineurin activity increased in P3 at 2 and 8 hours, respectively, returning to baseline at 24 hours. Nuclear factor of activated T cells (NFAT) nuclear translocation was enhanced in P3 cells. Ca2+-dependent signaling was probed with inhibitors of Ca2+/calmodulin (W-7), calcineurin (FK-506), and NFAT (INCA6): each prevented ICaL downregulation. Significant APD reductions (≈30%) at 24 hours in P3 cells were prevented by nimodipine, BAPTA-AM, W-7, or FK-506. Thus, rapid atrial cardiomyocyte activation causes Ca2+ loading, which activates the Ca2+-dependent calmodulin–calcineurin–NFAT system to cause transcriptional downregulation of ICaL, restoring Ca2+i to normal at the cost of APD reduction. These studies elucidate for the first time the molecular feedback mechanisms underlying arrhythmogenic AT remodeling.

Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors.

Risk factors for cardiovascular diseases (CVD) increase oxidative stress, and they are proposed to hasten endothelial cell (EC) damage and dysfunction. Our objective was to elucidate the impact of chronic exposure to risk factors for CVD on senescence of EC isolated and cultured from internal mammary arterial segments of patients with severe coronary artery disease. Senescence induced by serial passages resulted in progressive telomere shortening, and short initial telomeres predicted early appearance of senescence in culture. Neither time course of senescence nor telomere length was age-dependent, suggesting that biological age, rather than chronological age, determined the dynamics. Senescence appeared earlier in patients with longer history of risk factor for CVD, and multivariate analysis suggested that hypertension hastened the onset of senescence. Risk factors for CVD override the effects of chronological aging likely by generating stress-dependent damage: senescent EC exhibited oxidative stress (increase in lipid peroxydation and caveolin-1 gene expression) and cell damage markers (loss of eNOS expression and increase in Cox2 mRNA, lower TRF1 protein level). Thus, cell senescence was triggered both by telomere-dependent and -independent pathways. In conclusion, chronic exposure to risk factors for CVD accelerated the development of endothelial senescence that could contribute to the pathogenesis of CVD.

Nuclear-delimited Angiotensin Receptor-mediated Signaling Regulates Cardiomyocyte Gene Expression

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [α-32P]UTP (e.g. 36.0 ± 6.0 cpm/ng of DNA control versus 246.4 ± 15.4 cpm/ng of DNA Ang-II, 390.1 ± 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 ± 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFκB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFκB mRNA levels at saturating concentrations with ∼2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca2+] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca2+ release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

Mechanisms Underlying Rate-Dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes

Transient outward K+ current (Ito) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac Ito and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. Ito was recorded with whole-cell patch clamp. The 3-Hz pacing reduced Ito by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by ≈30% and ≈40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced Ito downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented Ito downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca2+i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na+/Ca2+ exchange and sarcoplasmic reticulum Ca2+-ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented Ito reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates Ito expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent Ito control.

Lung structural remodeling and pulmonary hypertension after myocardial infarction: complete reversal with irbesartan

OBJECTIVES The severity of pulmonary hypertension associated with heart failure carries a poor prognosis. The lungs are very sensitive to the constrictive and proliferative effects of angiotensin-II and could represent a preferential target for this peptide. METHODS Rats with large myocardial infarcts or sham surgery received the angiotensin-II receptor antagonist irbesartan (40 mg/kg/day) or vehicle for 2 or 8 weeks (n=5 to 8 for each group). Hemodynamic and morphometric measurements were obtained followed by immunohistochemistry, immunofluorescence analysis and electron microscopic characterization of lung sections. RESULTS The infarct groups developed progressive pulmonary hypertension and right ventricular hypertrophy with elevated left ventricular filling pressures (all P<0.01). Despite similar infarct size, filling pressures were lower (P<0.01) while pulmonary hypertension and right ventricular hypertrophy were completely normalized by irbesartan. Isolated lungs pressure-flow relationships were identical at 2 weeks. At 8 weeks it was steepest and shifted upward in the infarct group (P<0.001), and completely normalized by irbesartan. Lung weight doubled after infarct with no evidence of pulmonary edema and was also normalized by irbesartan. Important lungs structural remodeling evidenced by collagen and reticulin deposition, thickening of the alveolar septa and proliferation of cells with ultrastructural characteristics of myofibroblasts (pericytes) were identified after infarct. CONCLUSIONS After large myocardial infarct there is important pulmonary structural remodeling in which myofibroblasts (pericytes) proliferation may play an important role. This initially protective mechanism against high filling pressures could eventually contribute to the development of pulmonary hypertension and right ventricular hypertrophy. Future studies are needed to determine if angiotensin-II directly modulates pulmonary remodeling after myocardial infarct.

Essential Role of Protein Kinase Cδ in Platelet Signaling, αIIbβ3 Activation, and Thromboxane A2 Release

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCδ, but not PKCα or PKCβ, is required for collagen-induced phospholipase C-dependent signaling, activation of αIIbβ3, and platelet aggregation. Analysis of PKCδ phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by αIIbβ3 outside-in signaling. Moreover, PKCδ triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A2, which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCδ signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A2.

Resident Nestin+ Neural-Like Cells and Fibers Are Detected in Normal and Damaged Rat Myocardium

The present study examined whether nestin+ neural-like stem cells detected in the scar tissue of rats 1 week after myocardial infarction (MI) were derived from bone marrow and/or were resident cells of the normal myocardium. Irradiated male Wistar rats transplanted with β-actin promoter-driven, green fluorescent protein (GFP)–labeled, unfractionated bone marrow cells were subjected to coronary artery ligation. Three weeks after MI, GFP-labeled bone marrow cells were detected in the infarct region, and a modest number were associated with nestin immunoreactivity. The paucity of GFP+/nestin+ cells in the scar tissue provided the impetus to explore whether neural-like stem cells were derived from cardiac tissue. Nestin mRNA and immunoreactivity were detected in normal rat myocardium, and transcript levels were increased in the damaged heart after MI. In primary-passage, cardiac tissue-derived neural cells, filamentous nestin staining was associated with a diffuse, cytoplasmic glial fibrillary acidic protein signal. Unexpectedly, in viable myocardium, numerous nestin+/glial fibrillary acidic protein+ fiberlike structures of varying length were detected and observed in close proximity to neurofilament-M+ fibers. The infarct region was likewise innervated, and the preponderance of neurofilament-M+ fibers appeared to be physically associated with nestin+ fiberlike structures. These data highlight the novel observation that the normal rat heart contained resident nestin+/glial fibrillary acidic protein+ neural-like stem cells, fiberlike structures, and nestin mRNA levels that were increased in response to myocardial ischemia. Cardiac tissue-derived neural stem cell migration to the infarct region and concomitant nestin+ fiberlike innervation represent obligatory events of reparative fibrosis in the damaged rat myocardium.