Jonathan Ledoux

TRPC3-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 non-selective 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 (αSMA) protein-expression in fibroblasts. Ca 2+ -removal and TRPC3-blockade suppressed extracellular-signal regulated kinase (ERK)-phosphorylation, and ERK-phosphorylation inhibition reduced fibroblast-proliferation. TRPC3-expression was upregulated in atria from AF-patients, goats with electrically-maintained AF and tachypacing-induced heart-failure dogs. TRPC3-knockdown (shRNA-based) decreased canine atrial-fibroblast proliferation. In left-atrial (LA) fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial-tachypacing, TRPC3 protein-expression, currents, ERK-phosphorylation and extracellular-matrix gene-expression were all significantly increased. In cultured LA-fibroblasts from AF-dogs, proliferation-rates, αSMA-expression and ERK-phosphorylation were increased, and suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF-atria; experimental micro-RNA-26 knockdown reproduced AF-induced TRPC3-upregulation and fibroblast-activation. MicroRNA-26 has Nuclear Factor of Activated T-cells (NFAT) binding-sites in the 59-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 Ca 2+ -influx that activates ERK-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-block prevents AF-substrate development in a dog model of electrically-maintained AF. TRPC3 likely plays an important role in AF-promoting fibroblast pathophysiology and is a novel potential therapeutic target.Background— Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-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 (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 &mgr;mol/L). Pyrazole-3 suppressed angiotensin II–induced Ca2+ influx, proliferation, and &agr;-smooth muscle actin protein expression in fibroblasts. Ca2+ 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, &agr;-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 Ca2+ 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.

Differential regulation of Ca2+-activated Cl− currents in rabbit arterial and portal vein smooth muscle cells by Ca2+-calmodulin-dependent kinase

1 Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types. 2 In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve. 3 In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+. 4 In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells. 5 The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.

Modulation of Ca2+-dependent Cl− channels by calcineurin in rabbit coronary arterial myocytes

The role of the Ca2+‐dependent phosphatase calcineurin (CaN) in the modulation of Ca2+‐dependent Cl‐ channels (ClCa) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin‐dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca2+ was increased. ClCa currents (ICl(Ca)) elicited by cell dialysis with fixed intracellular Ca2+ levels up to 500 nm were inhibited by 10 μm cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage‐dependent manner, whereas currents evoked by 1 μm Ca2+ were not affected. Inhibition of CaN with CsA also led to a significant reduction in Ca2+ sensitivity of the channel at +50 mV; half‐maximal activation increased from 363 ± 16 nm Ca2+ in control to 515 ± 40 nm Ca2+ in the presence of CsA. Similar effects were observed on ICl(Ca) when a specific peptide fragment inhibitor of CaN (CaN‐AF, 5 μm) was dialysed into the cell via the pipette (500 nm Ca2+). Application of KN‐93 (10 μm), a specific inhibitor of CaMKII, enhanced ICl(Ca) in myocytes dialysed with 1 μm Ca2+ but produced no significant effect on this current when the cells were dialysed with 350 or 500 nm Ca2+. These results are consistent with the notion that in coronary arterial cells, the activity of ClCa is enhanced by dephosphorylation of the channel or a closely associated regulatory protein. Moreover the balance of CaN and CaMKII regulating ICl(Ca) is dependent on the level of Ca2+ used to activate ICl(Ca).

Increased peripheral resistance in heart failure: new evidence suggests an alteration in vascular smooth muscle function.

Increased peripheral resistance is a hallmark of chronic heart failure and has been primarily attributed to neurohumoral pathways involving both the renin–angiotensin and sympathetic nervous systems. The increased resistance is thought to serve as a compensatory mechanism to help maintain perfusion to the vital organs by sustaining blood pressure in the fate of a failing heart. Local mechanisms, and in particular endothelial dysfunction, have also been shown to be important contributors in regulating arterial resistance and vascular remodeling in this disease. In this issue of the British Journal of Pharmacology, Gschwend et al. (2003) present new data suggesting that in the absence of a functional endothelium, myogenic constriction of small pressurized mesenteric arteries, an intrinsic property of vascular smooth muscle cells, is enhanced in a coronary artery ligation‐induced myocardial infarction model of congestive heart failure (CHF) in the rat. The increased myogenic tone appears to be tightly linked to angiotensin II type 1 receptors (AT1). The possibility that CHF‐induced stimulation of myogenic constriction is due to the local release of preformed angiotensin II or constitutive upregulation of the AT1 receptor signaling pathways are discussed along with other potential cellular and molecular mechanisms previously suggested to play a role in myogenic reactivity.

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.