Nicholas Stafford

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes.

Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4(mut)GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4(mut)GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4(mut)GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart.

The plasma membrane calcium ATPases and their role as major new players in human disease.

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease.

The oxoglutarate receptor 1 (OXGR1) modulates pressure overload-induced cardiac hypertrophy in mice

The G-protein-coupled receptors (GPCRs) family of proteins play essential roles in the heart, including in the regulation of cardiac hypertrophy. One member of this family, the oxoglutarate receptor 1 (OXGR1), may have a crucial role in the heart because it acts as a receptor for α-ketoglutarate, a metabolite that is elevated in heart failure patients. OXGR1 is expressed in the heart but its precise function during cardiac pathophysiological process is unknown. Here we used both in vivo and in vitro approaches to investigate the role of OXGR1 in cardiac hypertrophy. Genetic ablation of Oxgr1 in mice (OXGR1−/−) resulted in a significant increase in hypertrophy following transverse aortic constriction (TAC). This was accompanied by reduction in contractile function as indicated by cardiac fractional shortening and ejection fraction. Conversely, adenoviral mediated overexpression of OXGR1 in neonatal rat cardiomyocytes significantly reduced phenylephrine-induced cardiomyocyte hypertrophy, a result that was consistent with the in vivo data. Using a combination of yeast two hybrid screening and phospho-antibody array analysis we identified novel interacting partner and downstream signalling pathway that might be regulated by the OXGR1. First, we found that OXGR1 forms a molecular complex with the COP9 signalosome complex subunit 5 (CSN5). Secondly, we observed that the STAT3 signalling pathway was upregulated in OXGR1−/− hearts. Since CSN5 interacts with TYK2, a major upstream regulator of STAT3, OXGR1 might regulate the pro-hypertrophic STAT3 pathway via interaction with the CSN5-TYK2 complex. In conclusion, our study has identified OXGR1 as a novel regulator of pathological hypertrophy via the regulation of the STAT3. Identification of molecules that can specifically activate or inhibit this receptor may be very useful in the development of novel therapeutic approach for pathological cardiac hypertrophy.

88 The effects of genetic ablation of microtubule-associated protein 1s (MAP1S) in regulating autophagy in the heart

Autophagy is an important process to maintain cellular homeostasis in many cell types including cardiomyocytes. In the heart, defective autophagy in response to pathological stimuli may lead to the development of adverse remodelling and eventually heart failure. The microtubule-associated protein 1S (MAP1S) has previously been identified as an interacting partner of the major autophagy regulator LC3; however, its role in the heart is not completely understood. In this study we investigated the role of microtubule-associated protein 1S (MAP1S) in regulating autophagy during a number of cardiac pathological conditions. siRNA gene silencing was used in Neonatal Rat Cardiomyocyte (NRCM) to knockdown MAP1S. The rate of autophagy was analysed using GFP-LC3 expressing adenovirus. This detection method was also used in fibroblast derived from MAP1S knock out mice (MAP1S -/-). Following rapamycin (5 uM) and chloroquine (3 uM) stimulation, both NRCM lacking MAP1S and Fibroblast derived from MAP1S knockout mice showed increased autophagy after 2 hours treatment compare to control. However, the expression level of p62 and beclin were not differ between NRCM lacking MAP1S, Fibroblast derived from MAP1S knockout mice and control. Interestingly, apoptosis as detected by TUNEL assay was significantly enhanced in NRCM lacking MAP1S. To test the effect of pathological stimuli, we subjected MAP1S-/- mice to transverse aortic constriction (TAC, 2 weeks) or myocardial infarction (MI, 4 weeks). Following MI, we found a significantly higher mortality in MAP1S-/- mice (60%) vs WT control (30%) although the extent of MI was comparable between MAP1S-/- and WT as indicated by cTnI level and the fibrotic infarct area. TUNEL assay exhibited higher apoptosis in MAP1S-/- mice which might contribute to the low survival rate. The surviving MAP1S-/- mice showed reduction in hypertrophic response and consistent in the pressure overload and MI model as we found lower HW/BW ratio in MAP1S-/- mice. This phenotype might be attributable to higher autophagy in the knockout animals. Our findings suggest that MAP1S modulates autophagy and apoptosis in cardiomyocytes. In vivo ablation of MAP1S might impair survival after MI and lead to an attenuated hypertrophic response following pressure overload.

The Control of Sub-plasma Membrane Calcium Signalling by the Plasma Membrane Calcium ATPase Pump PMCA4

Within cardiomyocytes cytosolic calcium levels rise and fall by an order of magnitude in each cardiac cycle, yet amidst the noise of this “global” calcium, a separate pool of “local” calcium is able to act as a second messenger in a multitude of signalling networks. The cell is equipped to deal with this through utilising the calcium-binding messenger protein calmodulin which in turn activates calcium/calmodulin-dependent targets and through compartmentalisation. This allows decoding of the calcium signal within such subcellular microdomains as the mitochondrion, the nucleus, the sarcoplasmic reticulum and the plasma membrane. In recent years our group and others have identified isoform 4 of the plasma membrane calcium/calmodulin-dependent ATPase (PMCA4) as a major regulator of local subplasmalemmal calcium in a number of cardiovascular cell types including the cardiomyocyte. Here we review techniques developed for the study of calcium levels local to PMCA4, the protein interaction and signalling complexes formed and regulated by the pump and the physiological implications of these in the heart and vascular systems.

176 Serotonin receptor 2b (5-ht2b) modulates cardiomyocyte proliferation by regulating the hippo pathway

Heart failure is one of the leading causes of death worldwide. In part, this is due to the inadequate regenerative capacity of cardiomyocytes post-injury. Modulation of the Hippo signalling pathway in mice has been shown to enhance cardiomyocyte proliferation and improve survival in a myocardial infarction model. While the discovery of the Hippo pathway and its function as a master regulator of cell proliferation has led to greater understanding of its core components such as Yes-associated protein (YAP), the upstream signals that regulate the Hippo pathway have remained elusive. This study was aimed to identify novel upstream regulators of the Hippo pathway in cardiomyocytes that could be targeted pharmacologically to induce regeneration. We performed a targeted RNAi screen in H9c2 cardiomyoblast cell line using adenovirus-mediated luciferase reporter system to detect the activity of YAP, the major effector of the Hippo pathway. Using this system, 5-hydroxytryptamine receptor 2B (5-HT2B) was identified as a potential regulator of the Hippo pathway. Serotonin-mediated 5-HT2B has previously been shown to play a significant role in cardiac development during embryogenesis; however, a link between 5-HT2B and the Hippo pathway has not yet been documented. An in vitro model was subsequently established by overexpressing 5-HT2B in primary neonatal rat cardiomyocytes (NRCMs) using an adenoviral system. The activities of different components of the Hippo pathway were investigated with an emphasis on YAP. Immunofluorescence microscopy was utilised to quantify cardiomyocyte proliferation and survival. Using this system, we found that overexpression of 5-HT2B in cardiomyocytes enhanced YAP activity by 12 folds compared to the control group as indicated by YAP-luciferase assay. In keeping with this, we observed an increase in YAP nuclear translocation following 5-HT2B overexpression, indicating YAP activation. Mechanistically, we found that 5-HT2B expression reduced Large tumour suppressor (LATS) phosphorylation, eventually leading to YAP activation. Since YAP is known to mediate cell proliferation we analysed proliferation rate in cardiomyocytes overexpressing 5-HT2B. We found that cell proliferation was increased by 39.7% compared with control cells as indicated by EdU incorporation assay. In conclusion, our findings have identified 5-HT2B as a novel upstream regulator of the Hippo pathway in cardiomyocytes. We also observed that in cardiomyocytes, 5-HT2B is a potent stimulator of YAP activity and cell proliferation. Since 5-HT2B is a membrane receptor that can be targeted pharmacologically, this finding may provide new insight for the development of a new approach to induce cardiomyocyte regeneration.