Wenbin Liang

Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18

The heartbeat originates within the sinoatrial node (SAN), a small structure containing <10,000 genuine pacemaker cells. If the SAN fails, the ∼5 billion working cardiomyocytes downstream of it become quiescent, leading to circulatory collapse in the absence of electronic pacemaker therapy. Here we demonstrate conversion of rodent cardiomyocytes to SAN cells in vitro and in vivo by expression of Tbx18, a gene critical for early SAN specification. Within days of in vivo Tbx18 transduction, 9.2% of transduced, ventricular cardiomyocytes develop spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. In vivo, focal Tbx18 gene transfer in the guinea-pig ventricle yields ectopic pacemaker activity, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquire the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells. The creation of induced SAN pacemaker (iSAN) cells opens new prospects for bioengineered pacemakers.

Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of the Myocardial N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Early Biomechanical Stress

Rationale: Mechanotransduction and the response to biomechanical stress is a fundamental response in heart disease. Loss of phosphoinositide 3-kinase (PI3K)&ggr;, the isoform linked to G protein–coupled receptor signaling, results in increased myocardial contractility, but the response to pressure overload is controversial. Objective: To characterize molecular and cellular responses of the PI3K&ggr; knockout (KO) mice to biomechanical stress. Methods and Results: In response to pressure overload, PI3K&ggr;KO mice deteriorated at an accelerated rate compared with wild-type mice despite increased basal myocardial contractility. These functional responses were associated with compromised phosphorylation of Akt and GSK-3&agr;. In contrast, isolated single cardiomyocytes from banded PI3K&ggr;KO mice maintained their hypercontractility, suggesting compromised interaction with the extracellular matrix as the primary defect in the banded PI3K&ggr;KO mice. &bgr;-Adrenergic stimulation increased cAMP levels with increased phosphorylation of CREB, leading to increased expression of cAMP-responsive matrix metalloproteinases (MMPs), MMP2, MT1-MMP, and MMP13 in cardiomyocytes and cardiofibroblasts. Loss of PI3K&ggr; resulted in increased cAMP levels with increased expression of MMP2, MT1-MMP, and MMP13 and increased MMP2 activation and collagenase activity in response to biomechanical stress. Selective loss of N-cadherin from the adhesion complexes in the PI3K&ggr;KO mice resulted in reduced cell adhesion. The &bgr;-blocker propranolol prevented the upregulation of MMPs, whereas MMP inhibition prevented the adverse remodeling with both therapies, preventing the functional deterioration in banded PI3K&ggr;KO mice. In banded wild-type mice, long-term propranolol prevented the adverse remodeling and systolic dysfunction with preservation of the N-cadherin levels. Conclusions: The enhanced propensity to develop heart failure in the PI3K&ggr;KO mice is attributable to a cAMP-dependent upregulation of MMP expression and activity and disorganization of the N-cadherin/&bgr;-catenin cell adhesion complex. &bgr;-Blocker therapy prevents these changes thereby providing a novel mechanism of action for these drugs.

Remote preconditioning provides potent cardioprotection via PI3K/Akt activation and is associated with nuclear accumulation of β-catenin.

rIPC [remote IPC (ischaemic preconditioning)] has been shown to invoke potent myocardial protection in animal studies and recent clinical trials. Although the important role of PI3K (phosphoinositide 3-kinase)/Akt activation in the cardioprotection afforded by local IPC is well described, our understanding of the intracellular signalling of rIPC remains incomplete. We therefore examined the hypothesis that the myocardial protection afforded by rIPC is mediated via the PI3K/Akt/GSK3β (glycogen synthase kinase 3β) signalling pathway, activation of which is associated with nuclear accumulation of β-catenin. rIPC was induced in mice using four cycles of 5 min of ischaemia and 5 min of reperfusion of the hindlimb using a torniquet. This led to reduced infarct size (19 ± 4% in rIPC compared with 39 ± 7% in sham; P<0.05), improved functional recovery and reduced apoptosis after global I/R (ischaemia/reperfusion) injury using a Langendorff-perfused mouse heart model. These effects were reversed by pre-treatment with an inhibitor of PI3K activity. Furthermore, Western blot analysis demonstrated that, compared with control, rIPC was associated with activation of the PI3K/Akt signalling pathway, resulting in phosphorylation and inactivation of GSK3β, accumulation of β-catenin in the cytosol and its translocation to the nucleus. Finally, rIPC increased the expression of β-catenin target genes involved in cell-survival signalling, including E-cadherin and PPARδ (peroxisome-proliferator-activated receptor δ). In conclusion, we show for the first time that the myocardial protection afforded by rIPC is mediated via the PI3K/Akt/GSK3β signalling pathway, activation of which is associated with nuclear accumulation of β-catenin and the up-regulation of its downstream targets E-cadherin and PPARδ involved in cell survival.

SHOX2 Overexpression Favors Differentiation of Embryonic Stem Cells into Cardiac Pacemaker Cells, Improving Biological Pacing Ability

Summary When pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of the native cardiac pacemaker (the sinoatrial node). Overexpression of SHOX2 during ESC differentiation upregulated the pacemaker gene program, resulting in enhanced automaticity in vitro and induced biological pacing upon transplantation in vivo. The accentuated automaticity is accompanied by temporally evolving changes in the effectors and regulators of Wnt signaling. Our findings provide a strategy for enriching the cardiac pacemaker cell population from ESCs.

Importance of cell-cell contact in the therapeutic benefits of cardiosphere-derived cells

Cardiosphere‐derived cells (CDCs) effect therapeutic regeneration after myocardial infarction (MI) both in animal models and in humans. Here, we test the hypothesis that cell‐cell contact plays a role in mediating the observed therapeutic benefits of CDCs, above and beyond conventional paracrine effects. Human CDCs or vehicle were injected into immunodeficient (SCID) mouse hearts during acute MI. CDC transplantation augmented the proportion of cycling (Ki67+) cardiomyocytes and improved ventricular function. CDC‐conditioned media only modestly augmented the percentage of Ki67+ cardiomyocytes (>control but

Regulation of Proliferation and Membrane Potential by Chloride Currents in Rat Pulmonary Artery Smooth Muscle Cells

Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to increased pulmonary vascular resistance and pulmonary hypertension. Because proliferation depends on membrane potential (Vm) and because Vm is, in part, determined by Cl− currents (ICl), we examined the effects of ICl inhibition with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on cultured rat PASMCs. DIDS (30 &mgr;mol/L) reduced cell numbers, decreased 5-bromodeoxyuridine incorporation and delayed cell cycle progression. ICl inhibition with 5-Nitro-2-(3-phenylpropylamino) benzoic acid (100 &mgr;mol/L) also reduced cell numbers of cultured rat PASMCs. To test the possible involvement of ICl in the regulation of PASMC proliferation, we measured Vm and ICl in both cultured (proliferating) and acutely dissociated (nonproliferating) rat PASMCs. Vm (−39.3±1.4 mV) was close to the equilibrium potential of Cl− (−39 mV) in proliferating PASMCs but differed from equilibrium potential of Cl− in acutely dissociated cells (−45.3±0.9 mV). DIDS and substitution of extracellular Cl− with I− induced Vm hyperpolarization in proliferating but not nonproliferating PASMCs. Consistent with Vm recordings, DIDS-sensitive baseline and swelling-activated (Ca2+-independent) ICls, recorded with low Ca2+ (<1 nmol/L) pipette solutions, were ≈5-fold greater in proliferating than in nonproliferating PASMCs. By contrast, Ca2+-activated ICl did not differ between proliferating and nonproliferating PASMCs. Ca2+-independent ICls were also increased in proliferating PASMCs acutely dissociated from rats exposed to hypoxia (10% O2; 7 days). These findings are consistent with the conclusion that ICls regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary hypertension.

Role of phosphoinositide 3-kinase {alpha}, protein kinase C, and L-type Ca2+ channels in mediating the complex actions of angiotensin II on mouse cardiac contractility.

Although angiotensin II (Ang II) plays an important role in heart disease associated with pump dysfunction, its direct effects on cardiac pump function remain controversial. We found that after Ang II infusion, the developed pressure and +dP/dtmax in isolated Langendorff-perfused mouse hearts showed a complex temporal response, with a rapid transient decrease followed by an increase above baseline. Similar time-dependent changes in cell shortening and L-type Ca2+ currents were observed in isolated ventricular myocytes. Previous studies have established that Ang II signaling involves phosphoinositide 3-kinases (PI3K). Dominant-negative inhibition of PI3K&agr; in the myocardium selectively eliminated the rapid negative inotropic action of Ang II (inhibited by ≈90%), whereas the loss of PI3K&ggr; had no effect on the response to Ang II. Consistent with a link between PI3K&agr; and protein kinase C (PKC), PKC inhibition (with GF 109203X) reduced the negative inotropic effects of Ang II by ≈50%. Although PI3K&agr; and PKC activities are associated with glycogen synthase kinase-3&bgr; and NADPH oxidase, genetic ablation of either glycogen synthase kinase-3&bgr; or p47phox (an essential subunit of NOX2-NADPH oxidase) had no effect on the inotropic actions of Ang II. Our results establish that Ang II has complex temporal effects on contractility and L-type Ca2+ channels in normal mouse myocardium, with the negative inotropic effects requiring PI3K&agr; and PKC activities.

Wnt signalling suppresses voltage-dependent Na⁺ channel expression in postnatal rat cardiomyocytes.

Wnt signalling is activated in arrhythmogenic heart diseases, but its role in the regulation of cardiac ion channel expression is unknown. Exposure of neonatal rat ventricular myocytes to Wnt3a, an activator of canonical Wnt signalling, decreases Scn5a mRNA, Nav1.5 protein and Na+ current density. Wnt3a does not affect the inward rectifier K+ current or L‐type Ca2+ channels. The Wnt pathway is a negative regulator of cardiac Na+ channel expression and may play a role in altered ion channel expression in heart disease.

L-Tryptophan ethyl ester dilates small mesenteric arteries by inhibition of voltage-operated calcium channels in smooth muscle

BACKGROUND AND PURPOSE L‐tryptophan (L‐W) is a precursor of the vasoconstrictor, 5‐HT. However, acute administration of L‐W ethyl ester (L‐Wee) lowered blood pressure. The mechanism of action is unknown. This study compares the vascular effects of L‐W and L‐Wee in intact animals, isolated aortic rings, small mesenteric arteries (MA) and explores possible mechanisms by studies in vascular smooth muscle cells (VSMC) of MA.

Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells.

Background: Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl− currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl− current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown. Methods and results: In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 &mgr;mol/l) ICl,swell and inhibited (IC50 = 6.9 &mgr;mol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl− channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl− channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution. Conclusion: Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.