Elizabeth A. Woodcock

β2-Adrenergic Receptor Overexpression Exacerbates Development of Heart Failure After Aortic Stenosis

Background—β-Adrenergic signaling is downregulated in the failing heart, and the significance of such change remains unclear. Methods and Results—To address the role of β-adrenergic dysfunction in heart failure (HF), aortic stenosis (AS) was induced in wild-type (WT) and transgenic (TG) mice with cardiac targeted overexpression of β2-adrenergic receptors (ARs), and animals were studied 9 weeks later. The extents of increase in systolic arterial pressure (P<0.01 versus controls), left ventricular (LV) hypertrophy (TG, 94±6 to 175±7 mg; WT, 110±6 to 168±10 mg; both P<0.01), and expression of ANP mRNA were similar between TG and WT mice with AS. TG mice had higher incidences of premature death and critical illness due to heart failure (75% versus 23%), pleural effusion (81% versus 45%), and left atrial thrombosis (81% versus 36%, all P<0.05). A more extensive focal fibrosis was found in the hypertrophied LV of TG mice (P<0.05). These findings indicate a more severe LV dysfunction in TG mice. In sham-operated...

Reduced Phosphoinositide 3-Kinase (p110α) Activation Increases the Susceptibility to Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110alpha) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110alpha) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult ( approximately 4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice ( approximately 15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110alpha) and AF.

Gq-initiated cardiomyocyte hypertrophy is mediated by phospholipase Cβ1b

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell culture models. Hypertrophic responses induced by pressure or volume overload are exacerbated by increased Gq activity and ameliorated by Gq inhibition. Gq activates phospholipase Cβ (PLCβ) subtypes, resulting in generation of the intracellular messengers inositol(1,4,5)tris‐phosphate [Ins(1,4,5)P3] and sn‐1,2‐diacylglycerol (DAG), which regulate intracellular Ca2+ and conventional protein kinase C subtypes, respectively. Gq can also signal independently of PLCβ, and the involvement of either Ins(1,4,5)P3 or DAG in cardiomyocyte hypertrophy has not been unequivocally established. Overexpression of one splice variant of PLCβ1, specifically PLCβ1b, in neonatal rat cardiomyocytes causes increased cell size, elevated protein/ DNA ratio, and heightened expression of the hypertrophy‐related marker gene, atrial natriuretic peptide. The other splice variant, PLCβ1a, had no effect. Expression of a 32‐aa C‐terminal PLCβ1b peptide, which competes with PLCβ1b for sarcolemmal association, prevented PLC activation and eliminated hypertrophic responses initiated by Gq or Gq‐coupled α1‐adrenergic receptors. In contrast, a PLCβ1a C‐terminal peptide altered neither PLC activity nor cellular hypertrophy. We conclude that hypertrophic responses initiated by Gq are mediated specifically by PLCβ1b. Preventing PLCβ1b association with the sarcolemma may provide a useful therapeutic target to limit hypertrophy.—Filtz, T. M., Grubb, D. R., McLeod‐Dryden, T. J., Luo, J., Woodcock, E. A. Gq‐initiated cardiomyocyte hypertrophy is mediated by phospholipase Cβ1b. FASEB J. 23, 3564–3570 (2009). www.fasebj.org

Phosphoinositide signalling and cardiac arrhythmias

Arrhythmias arise from a complex interaction between structural changes in the myocardium and changes in cellular electrophysiology. Electrophysiological balance requires precise control of sarcolemmal ion channels and exchangers, many of which are regulated by phospholipid, phosphatidylinositol(4,5)bisphosphate. Phosphatidylinositol(4,5)bisphosphate is the immediate precursor of inositol(1,4,5)trisphosphate, a regulator of intracellular Ca2+ signalling and, therefore, a potential contributor to arrhythmogenesis by altering Ca2+ homeostasis. The aim of the present review is to outline current evidence that this signalling pathway can be a player in the initiation or maintenance of arrhythmias.

Distribution and Functional Role of Inositol 1,4,5-trisphosphate Receptors in Mouse Sinoatrial Node

Rationale: Inositol 1,4,5-trisphosphate receptors (IP3Rs) have been implicated in the generation of arrhythmias and cardiac muscle nuclear signaling. However, in the mammalian sinoatrial node (SAN), where the heart beat originates, the expression and functional activity of IP3Rs have not been investigated. Objectives: To determine whether SAN express IP3Rs and which isoforms are present. To examine the response of the SAN to IP3R agonists and antagonist, and the potential role played by IP3Rs in cardiac pacemaking. Methods and Results: The expression and distribution of IP3Rs were studied by reverse-transcription polymerase chain reaction, Western blotting, and immunolabeling. Ca2+ signaling and electric activity in intact mouse SAN were measured with Ca2+-sensitive fluorescent dyes. We found that although the entire SAN expressed three IP3R mRNA isoforms, the type II IP3R (IP3R2) was the predominant protein isoform detected by Western blot using protein extracts from the SAN, atrioventricular node, and atrial tissue. Immunohistochemistry studies also showed that IP3R2 was expressed in the central SAN region. Studies using isolated single pacemaker cells revealed that IP3R2 (but not IP3R1) was located with a similar distribution to the sarcoplasmic reticulum marker protein SERCA2a with some labeling adjacent to the surface membrane. The application of membrane-permeable IP3 (IP3-butyryloxymethyl ester) increased Ca2+ spark frequency and the pacemaker firing rate in single isolated pacemaker cells. In intact SAN preparations, IP3R agonists, endothelin-1 and IP3-butyryloxymethyl ester both increased intracellular Ca2+ and the pacemaker firing rate, whereas the IP3R antagonist, 2-aminoethoxydiphenyl borate decreased Ca2+ and the firing rate. Both of these effects were absent in the SAN from transgenic IP3R2 knockout mice. Conclusions: This study provides new evidence that functional IP3R2s are expressed in the mouse SAN and could serve as an additional Ca2+-dependent mechanism in modulating cardiac pacemaker activity as well as other Ca2+-dependent processes.

Selective activation of the “b” splice variant of phospholipase Cβ1 in chronically dilated human and mouse atria

Atrial fibrillation (AF) is commonly associated with chronic dilatation of the left atrium, both in human disease and animal models. The immediate signaling enzyme phospholipase C (PLC) is activated by mechanical stretch to generate the Ca2+-releasing messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol (DAG), an activator of protein kinase C subtypes. There is also evidence that heightened activity of PLC, caused by the receptor coupling protein Gq, can contribute to atrial remodelling. We examined PLC activation in right and left atrial appendage from patients with mitral valve disease (VHD) and in a mouse model of dilated cardiomyopathy caused by transgenic overexpression of the stress-activated protein kinase, mammalian sterile 20 like kinase 1 (Mst1) (Mst1-TG). PLC activation was heightened 6- to 10-fold in atria from VHD patients compared with right atrial tissue from patients undergoing coronary artery bypass surgery (CABG) and was also heightened in the dilated atria from Mst1-TG. PLC activation in human left atrial appendage and in mouse left atria correlated with left atrial size, implying a relationship between PLC activation and chronic dilatation. Dilated atria from human and mouse showed heightened expression of PLCbeta1b, but not of other PLC subtypes. PLCbeta1b, but not PLCbeta1a, caused apoptosis when overexpressed in neonatal rat cardiomyocytes, suggesting that PLCbeta1b may contribute to chamber dilatation. The activation of PLCbeta1b is a possible therapeutic target to limit atrial remodelling in VHD patients.

Scaffolding protein Homer 1c mediates hypertrophic responses downstream of Gq in cardiomyocytes

Activation of the heterotrimeric G protein, Gq, causes cardiomyocyte hypertrophy in vivo and in cell models. Responses to activated Gq in cardiomyocytes are mediated exclusively by phospholipase Cβ1b (PLCβ1b), because it localizes at the sarcolemma by binding to Shank3, a high‐molecular‐weight (MW) scaffolding protein. Shank3 can bind to the Homer family of low‐MW scaffolding proteins that fine tune Ca2+ signaling by facilitating crosstalk between Ca2+ channels at the cell surface with those on intracellular Ca2+ stores. Activation of α1‐adrenergic receptors, expression of constitutively active Gαq (GαqQL), or PLCβ1b initiated cardiomyocyte hypertrophy and increased Homer 1c mRNA expression, by 1.6 ± 0.18‐, 1.9 ± 0.17‐, and 1.5 ± 0.07‐fold, respectively (means ± SE, 6 independent experiments, P<0.05). Expression of Homer 1c induced an increase in cardiomyocyte area from 853 ± 27 to 1146 ± 31 μm2 (P<0.05); furthermore, expression of dominant‐negative Homer (Homer 1a) reversed the increase in cell size caused by α1‐adrenergic agonist or PLCβ1b treatment (1503±48 to 996±28 and 1626±48 to 828±31 (μm2, respectively, P<0.05). Homer proteins were localized near the sarcolemma, associated with Shank3 and phospholipase Cβ1b. We conclude that Gq‐mediated hypertrophy involves activation of PLCβ1b scaffolded onto a Shank3/Homer complex. Signaling downstream of Homer 1c is necessary and sufficient for Gq‐initiated hypertrophy.—Grubb, D. R., Luo, J., Yu, Y. L., Woodcock, E. A. Scaffolding protein Homer 1c mediates hypertrophic responses downstream of Gq in cardiomyocytes. FASEB J. 26, 596–603 (2012). www.fasebj.org

Inositol 1,4,5-trisphosphate receptors and pacemaker rhythms

Intracellular Ca(2+) plays an important role in the control of the heart rate through the interaction between Ca(2+) release by ryanodine receptors in the sarcoplasmic reticulum (SR) and the extrusion of Ca(2+) by the sodium-calcium exchanger which generates an inward current. A second type of SR Ca(2+) release channel, the inositol 1,4,5-trisphosphate receptor (IP(3)R), can release Ca(2+) from SR stores in many cell types, including cardiac myocytes. However, it is still uncertain whether IP(3)Rs play any functional role in regulating the heart rate. Accumulated evidence shows that IP(3) and IP(3)R are involved in rhythm control in non-cardiac pacemaker tissues and in the embryonic heart. In this review we focus on intracellular Ca(2+) oscillations generated by Ca(2+) release from IP(3)R that initiates membrane depolarization and provides a common mechanism producing spontaneous activity in a range of cells with pacemaker function. Emerging new evidence also suggests that IP(3)/IP(3)Rs play a functional role in normal and diseased hearts and in cardiac rhythm control. Several membrane currents, including a store-operated Ca(2+) current, might be activated by Ca(2+) release from IP(3)Rs. IP(3)/IP(3)R may thus add another dimension to the complex regulation of heart rate.

Diverse regulation of cardiac expression of relaxin receptor by α1- and β 1-adrenoceptors

PurposeRelaxin, a new drug for heart failure therapy, exerts its cardiac actions through relaxin family peptide receptor 1 (RXFP1). Factors regulating RXFP1 expression remain unknown. We have investigated effects of activation of adrenoceptors (AR), an important modulator in the development and prognosis of heart failure, on expression of RXFP1 in rat cardiomyocytes and mouse left ventricles (LV).MethodsExpression of RXFP1 at mRNA (real-time PCR) and protein levels (immunoblotting) was measured in cardiomyocytes treated with α- and β-AR agonists or antagonists. RXFP1 expression was also determined in the LV of transgenic mouse strains with cardiac-restricted overexpression of α1A-, α1B- or β2-AR. Specific inhibitors were used to explore signal pathways involved in α1-AR mediated regulation of RXFP1 in cardiomyocytes.ResultsIn cultured cardiomyocytes, α1-AR stimulation resulted in 2–3 fold increase in RXFP1 mRNA (P < 0.001), which was blocked by specific inhibitors for protein kinase C (PKC) or mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK). Activation of β1-, but not β2-AR, significantly inhibited RXFP1 expression (P < 0.001). Relative to respective wild-type controls, RXFP1 mRNA levels in the LV of mice overexpressing α1A- or α1B-AR were increased by 3- or 10-fold, respectively, but unchanged in β2-AR transgenic hearts. Upregulation by α1-AR stimulation RXFP1 expression was confirmed at protein levels both in vitro and in vivo.ConclusionsExpression of RXFP1 was up-regulated by α1-AR but suppressed by β-AR, mainly β1-AR subtype, in cardiomyocytes. Future studies are warranted to characterize the functional significance of such regulation, especially in the setting of heart failure.

No Contribution of IP3-R(2) to Disease Phenotype in Models of Dilated Cardiomyopathy or Pressure Overload Hypertrophy

Background—We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20–like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. Methods and Results—To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)−/−) had been deleted (DCM-2Tg×IP3-R(2)−/−) and transverse aortic constriction was performed on IP3-R(2)−/− mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)−/− were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. Conclusions—We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.