Vascular CaMKII: heart and brain in your arteries.

First characterized in neuronal tissues, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) is a key signaling component in several mammalian biological systems. Its unique capacity to integrate various Ca(2+) signals into different specific outcomes is a precious asset to excitable and nonexcitable cells. Numerous studies have reported roles and mechanisms involving CaMKII in brain and heart tissues. However, corresponding functions in vascular cell types (endothelium and vascular smooth muscle cells) remained largely unexplored until recently. Investigation of the intracellular Ca(2+) dynamics, their impact on vascular cell function, the regulatory processes involved and more recently the spatially restricted oscillatory Ca(2+) signals and microdomains triggered significant interest towards proteins like CaMKII. Heteromultimerization of CaMKII isoforms (four isoforms and several splice variants) expands this kinase's peculiar capacity to decipher Ca(2+) signals and initiate specific signaling processes, and thus controlling cellular functions. The physiological functions that rely on CaMKII are unsurprisingly diverse, ranging from regulating contractile state and cellular proliferation to Ca(2+) homeostasis and cellular permeability. This review will focus on emerging evidence of CaMKII as an essential component of the vascular system, with a focus on the kinase isoform/splice variants and cellular system studied.

Feed the brain: insights into the study of neurovascular coupling.

The microcirculation is tightly regulated by a diverse range of mechanisms which share the common goal of matching blood flow delivery with tissue metabolic demand. Despite in-depth examination of tissues like skeletal muscle, brain microcirculation has remained largely unexplored due to methodological limitations. Recent, technological advances have, however, started to grant greater access to this vital microcirculatory bed. This overview is part of a Special Topics Issue centered on the methodology, theory, and mechanistic basis of neurovascular coupling. Solicited manuscripts have been purposely written in an opinionated manner to provoke thought and to illuminate new emerging areas of investigation.

Transient receptor potential canonical-3 channel-dependent fibroblast regulation in atrial fibrillation.

BACKGROUND: Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca(2+)-permeable transient receptor potential canonical-3 (TRPC3) channels. METHODS AND RESULTS: Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (I(NSC)) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 µmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca(2+) influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca(2+) removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5' promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. CONCLUSIONS: TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca(2+) influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

Endothelial histamine H1 receptor signaling reduces blood-brain barrier permeability and susceptibility to autoimmune encephalomyelitis.

Disruption of the blood-brain barrier (BBB) underlies the development of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Environmental factors, such as Bordetella pertussis, are thought to sensitize central endothelium to biogenic amines like histamine, thereby leading to increased BBB permeability. B. pertussis-induced histamine sensitization (Bphs) is a monogenic intermediate phenotype of EAE controlled by histamine H(1) receptor (Hrh1/H(1)R). Here, we transgenically overexpressed H(1)R in endothelial cells of Hrh1-KO (H(1)RKO) mice to test the role of endothelial H(1)R directly in Bphs and EAE. Unexpectedly, transgenic H(1)RKO mice expressing endothelial H(1)R under control of the von Willebrand factor promoter (H(1)RKO-vWF(H1R) Tg) were Bphs-resistant. Moreover, H(1)RKO-vWF(H1R) Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H(1)RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H(1)R expression reduces BBB permeability, suggesting that endothelial H(1)R signaling may be important in the maintenance of cerebrovascular integrity.

Adenylate cyclase type 9 antagonizes cAMP accumulation and regulates endothelial signalling involved in atheroprotection.

AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.

Automated region of interest analysis of dynamic Ca²+ signals in image sequences.

Ca(2+) signals are commonly measured using fluorescent Ca(2+) indicators and microscopy techniques, but manual analysis of Ca(2+) measurements is time consuming and subject to bias. Automated region of interest (ROI) detection algorithms have been employed for identification of Ca(2+) signals in one-dimensional line scan images, but currently there is no process to integrate acquisition and analysis of ROIs within two-dimensional time lapse image sequences. Therefore we devised a novel algorithm for rapid ROI identification and measurement based on the analysis of best-fit ellipses assigned to signals within noise-filtered image sequences. This algorithm was implemented as a plugin for ImageJ software (National Institutes of Health, Bethesda, MD). We evaluated the ability of our algorithm to detect synthetic Gaussian signal pulses embedded in background noise. The algorithm placed ROIs very near to the center of a range of signal pulses, resulting in mean signal amplitude measurements of 99.06 ± 4.11% of true amplitude values. As a practical application, we evaluated both agonist-induced Ca(2+) responses in cultured endothelial cell monolayers, and subtle basal endothelial Ca(2+) dynamics in opened artery preparations. Our algorithm enabled comprehensive measurement of individual and localized cellular responses within cultured cell monolayers. It also accurately identified characteristic Ca(2+) transients, or Ca(2+) pulsars, within the endothelium of intact mouse mesenteric arteries and revealed the distribution of this basal Ca(2+) signal modality to be non-Gaussian with respect to amplitude, duration, and spatial spread. We propose that large-scale statistical evaluations made possible by our algorithm will lead to a more efficient and complete characterization of physiologic Ca(2+)-dependent signaling.

Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.

Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP](i). Regulatory fragments of PKG I alpha, PKG I beta, and an N-terminal deletion mutant of PKG I alpha were fused to circular permutated EGFP to generate alpha-, beta-, and delta-FlincG, with high dynamic ranges and apparent K(D,cGMP) values of 35 nM, 1.1 microM, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. delta-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, delta-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP](i) in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, delta-FlincG had an EC(50,cGMP) of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC(50,DEA/NO) = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP](i), which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets.

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.

A Variant in the Nicotinic Acetylcholine Receptor Alpha 3 Subunit Gene Is Associated With Hypertension Risks in Hypogonadic Patients.

Ephb6 gene knockout causes hypertension in castrated mice. EPHB6 controls catecholamine secretion by adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent way. Nicotinic acetylcholine receptor (nAChR) is a ligand-gated Ca2+/Na+ channel, and its opening is the first signaling event leading to catecholamine secretion by AGCCs. There is a possibility that nAChR might be involved in EPHB6 signaling, and thus sequence variants of its subunit genes are associated with hypertension risks. CHRNA3 is the major subunit of nAChR used in human and mouse AGCCs. We conducted a human genetic study to assess the association of CHRNA3 variants with hypertension risks in hypogonadic males. The study cohort included 1,500 hypogonadic Chinese males with (750 patients) or without (750 patients) hypertension. The result revealed that SNV rs3743076 in the fourth intron of CHRNA3 was significantly associated with hypertension risks in the hypogonadic males. We further showed that EPHB6 physically interacted with CHRNA3 in AGCCs, providing a molecular basis for nAChR being in the EPHB6 signaling pathway.

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium.

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.

Echocardiographic validation of pulmonary hypertension due to heart failure with reduced ejection fraction in mice.

Pulmonary hypertension (PH) associated with left heart diseases is the most prevalent cause of PH. The scarcity of studies exploring the pathophysiology and therapies of group II PH resides in the lack of validated small animal models with non-invasive determination of the presence and severity of PH. Heart failure (HF) was induced in mice by coronary artery ligation. Mice developed PH as evidenced by an elevated right ventricular (RV) systolic pressure and RV hypertrophy. Detailed non-invasive echocardiographic analysis on the left and right ventricles showed impaired left ventricular (LV) systolic and diastolic function. In addition, RV hypertrophy was confirmed by echo and accompanied by impaired function as well as increased pulmonary resistance. Correlation analysis validated the use of the LV wall-motion score index (WMSI) at a threshold value of ≥2.0 as a powerful and reliable indicator for the presence of PH and RV dysfunction. Echocardiography is an accurate non-invasive technique to diagnose PH in a HF mouse model. Moreover, an echocardiographic parameter of infarct size and LV function, the LV WMSI, reliably correlates with the presence of PH, RV hypertrophy and RV dysfunction and could be used to improve efficiency and design of pre-clinical studies.

EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells.

Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.

Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells.

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-ß1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-ß1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility.

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Lung Capillary Stress Failure and Arteriolar Remodelling in Pulmonary Hypertension Associated with Left Heart Disease (Group 2 PH).

Left heart diseases (LHD) represent the most prevalent cause of pulmonary hypertension (PH), yet there are still no approved therapies that selectively target the pulmonary circulation in LHD. The increase in pulmonary capillary pressure due to LHD is a triggering event leading to physical and biological alterations of the pulmonary circulation. Acutely, mechanosensitive endothelial dysfunction and increased capillary permeability combined with reduced fluid resorption lead to the development of interstitial and alveolar oedema. From repeated cycles of such capillary stress failure originate more profound changes with pulmonary endothelial dysfunction causing increased basal and reactive pulmonary vascular tone. This contributes to pulmonary vascular remodelling with increased arterial wall thickness, but most prominently, to alveolar wall remodelling characterized by myofibroblasts proliferation with collagen and interstitial matrix deposition. Although protective against acute pulmonary oedema, alveolar wall thickening becomes maladaptive and is responsible for the development of a restrictive lung syndrome and impaired gas exchanges contributing to shortness of breath and PH. Increasing awareness of these processes is unraveling novel pathophysiologic processes that could represent selective therapeutic targets. Thus, the roles of caveolins, of the intermediate myofilament nestin and of endothelial calcium dyshomeostasis were recently evaluated in pre-clinical models. The pathophysiology of PH due to LHD (group II PH) is distinctive from other groups of PH. Therefore, therapies targeting PH due to LHD must be evaluated in that context.

Single-cell microinjection coupled to confocal microscopy to characterize nuclear membrane receptors in freshly isolated cardiomyocytes.

Lipid bilayers, such as the plasma membrane and nuclear envelope, serve as effective cellular barriers to ions and macromolecules, thus allowing regulated access to subcellular compartments including the cytoplasm and nucleus, respectively. Of course, these barriers are semipermeable and a wide variety of proteins including transporters, ion exchangers, pumps, and ion channels are required to permit access as well as establish and maintain molecular and ionic gradients across membranes. However, some experimental designs, such as specifically targeting intracellular receptors, require the administration of membrane-impermeable molecules directly into live cells. The microinjection technique described in this chapter is an efficient, technically simple, and reliable approach that can be used to introduce macromolecules into intracellular compartments while maintaining the integrity of the plasma membrane itself.

Lymphatic network in atherosclerosis: the underestimated path.

The lymphatic system is a key component of tissue fluid homeostasis. In contrast to the closed and high-pressure blood vascular system, the lymphatic vascular system transports lymph in an open and low-pressure network. A prerequisite player in the transport of immune cells and cholesterol metabolism, it has been understudied until recently. Whereas defects in lymph circulation are mostly associated with pathologies such as congenital or acquired lymphedema, emerging significant developments are unraveling the role of lymphatic vessels in other pathological settings. In the last decade, discoveries of underlying genes responsible for developmental and postnatal lymphatic growth, combined with state-of-the-art lymphatic function imaging and quantification techniques, have matched the growing interest in understanding the role of the lymphatic system in atherosclerosis. With a historical perspective, this review highlights the current knowledge regarding interaction between the lymphatic vascular tree and atherosclerosis, with an emphasis on the physiological mechanisms of this multifaceted system throughout disease onset and progression.

CaMKII regulates intracellular Ca²âº dynamics in native endothelial cells.

Localized endothelial Ca(2+) signalling, such as Ca(2+) pulsars, can modulate the contractile state of the underlying vascular smooth muscle cell through specific endothelial targets. In addition to K(Ca)3.1 as a target, Ca(2+) pulsars, an IP3R-dependent pulsatile Ca(2+) release from the endoplasmic reticulum (ER) could activate a frequency-sensitive Ca(2+)-dependent kinase such as CaMKII. In the absence of extracellular Ca(2+), acetylcholine increased endothelial CaMKII phosphorylation and activation, thereby suggesting CaMKII activation independently of Ca(2+) influx. Herein, a reciprocal relation where CaMKII controls endothelial Ca(2+) dynamics has been investigated in mesenteric arteries. Both CaMKIIα and ß isoforms have been identified in endothelial cells and close proximity (<40 nm) suggests their association in heteromultimers. Intracellular Ca(2+) monitoring with high speed confocal microscopy then showed that inhibition of CaMKII with KN-93 significantly increased the population of Ca(2+) pulsars active sites (+89%), suggesting CaMKII as a major regulator of Ca(2+) pulsars in native endothelium. Mechanistic insights were then sought through the elucidation of the impact of CaMKII on ER Ca(2+) store. ER Ca(2+) emptying was accelerated by CaMKII inhibition and ER Ca(2+) content was assessed using ionomycin. Exposure to KN-93 strongly diminished ER Ca(2+) content (-61%) by relieving CaMKII-dependent inhibition of IP3 receptors (IP3R). Moreover, in situ proximity ligation assay suggested CaMKII-IP3R promiscuity, essential condition for a protein-protein interaction. Interestingly, segregation of IP3R within myoendothelial projection (MEP) appears to be isoform-specific. Hence, only IP3R type 1 and type 2 are detected within fenestrations of the internal elastic lamina, sites of MEP, whilst type 3 is absent from these structures. In summary, CaMKII seems to act as a Ca(2+)-sensitive switch of a negative feedback loop regulating endothelial Ca(2+) homeostasis, including Ca(2+) pulsars.

Spatiotemporal stability of neonatal rat cardiomyocyte monolayers spontaneous activity is dependent on the culture substrate.

In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS) is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-ß, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and ß2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (ß3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.