Changes in cardiomyocyte structure and cAMP/cGMP signalling during heart failure

The contractile function of the heart depends on efficient β adrenergic receptor (βAR) signalling which involves cycling nucleotides as second messengers. Correct secondary messenger signalling is only possible in healthy, well structured cardiac myocytes. Of the three βAR subtypes present in human cardiomyocytes β1AR and β2AR classically signal via 3'-5' cyclic adenosine monophosphate (cAMP) to regulate contraction after catecholamine administration, whereby the second isoform may also be cardioprotective. The far less characterised β3AR has been controversially associated to both increasing contraction through cAMP and protecting the heart through 3'-5' cyclic guanosine monophosphate (cGMP) signalling. During the progression of heart failure following myocardial infarction (MI) both the normal cell structure and the regulation of cAMP and cGMP signalling are changed. This happens in part due to changes in catecholaminergic stimulation of the βARs and in mechanical load, as well as due to a progressive development of hypertrophy. Some of the alterations initially appear to be of a compensatory nature but escalate into HF by worsening cardiomyocyte function and cell survival. The work presented here (1) investigates the structural integrity of healthy, isolated, single cardiomyocytes by looking at the surface topography via Scanning Ion Conductance Microscopy (SICM) imaging and by examining the internal Transverse Axial Tubule (TAT) network via confocal imaging; (2) elucidates the cyclic nucleotide response to catecholamine stimulation following either global (in the solution) or local (in the SICM pipette) stimulation of either β2ARs or β3ARs and measuring either cAMP or cGMP levels via Förster Resonance Energy Transfer (FRET) sensors in a combined FRET/SICM imaging setup; (3) determines how both the structure and β2AR and β3AR dependent second messenger signalling change in a progressive rat model of HF 4, 8 and 16 weeks after the induction of chronic MI. The major findings of the presented work are as follows: In control cardiomyocytes the structure is highly intricate with regular Z-grooves and crest areas. In MI cells the normal suface topography progressively deteriorates, with the eventual disappearance of Z-grooves by week 16, which correlates with the disorganisation of the cardiomyocyte’s internal transverse axial tubule (TAT) network of T-tubules emanating from the cell surface and traversing into the cell centre. This is accompanied by the gradual redistribution of β2ARs from their normal position inside the T-tubules to the unstructured areas on the cardiomyocyte membrane. The regularity and density of the TAT network is already severely compromised at 4 weeks post MI; at the same time a significant drop in the expression of the structural protein Junctophilin 2 (JPH2) occurs. At 4 and 8 weeks post MI a potentially compensatory increase in the number of longitudinal elements takes place which was no longer detectable at 16 weeks. The production of cAMP following local stimulation of β2ARs in the T-tubule openings was already suppressed at 4 weeks post MI and a β2AR response becomes detectable after local stimulation at the cell crests (areas between Z-grooves) at 8 weeks post MI. At 16 weeks post MI the β2AR-dependent cAMP level following both global and local stimulations was reduced due to an overall decrease in the adenylate cyclase (AC) activity. The production of the second cyclic nucleotide, cGMP, following β3AR stimulation is evident in control cells and to a significantly lesser extent in myocytes isolated from hearts at the end stage of HF. These β3AR-cGMP levels were degraded mainly by phosphodiesterases (PDE) 2 and 5. Local stimulation through the SICM pipette reveals that functional β3ARs are primarily localized inside T-tubules in control cells but redistribute equally in between T-tubules and crests in cells isolated from failing hearts. To improve the accuracy and reliability of local application of agonists via the SICM nanopipette voltage was applied to the pipette, as opposed to previously employed displacement of the liquid in the pipette via air pressure. Mathematical modelling served to determine the correct settings for this voltage driven application. It shows that the SICM nanopipette can reliably and precisely unload the βAR agonist ISO onto the nanoscale structure of cardiomyocytes via voltage.Open Acces

Shape and Compliance of Endothelial Cells after Shear Stress In Vitro or from Different Aortic Regions: Scanning Ion Conductance Microscopy Study

Objective: To measure the elongation and compliance of endothelial cells subjected to different patterns of shear stress in vitro, and to compare these parameters with the elongation and compliance of endothelial cells from different regions of the intact aorta. Materials and Methods: Porcine aortic endothelial cells were cultured for 6 days under static conditions or on an orbital shaker. The shaker generated a wave of medium, inducing pulsatile shear stress with a preferred orientation at the edge of the well or steadier shear stress with changing orientation at its centre. The topography and compliance of these cells and cells from the inner and outer curvature of ex vivo porcine aortic arches were measured by scanning ion conductance microscopy (SICM). Results: Cells cultured under oriented shear stress were more elongated and less compliant than cells grown under static conditions or under shear stress with no preferred orientation. Cells from the outer curvature of the aorta were more elongated and less compliant than cells from the inner curvature. Conclusion: The elongation and compliance of cultured endothelial cells vary according to the pattern of applied shear stress, and are inversely correlated. A similar inverse correlation occurs in the aortic arch, with variation between region

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Studying GPCR/cAMP pharmacology from the perspective of cellular structure

Signal transduction via G-protein coupled receptors (GPCRs) relies upon the production of cAMP and other signaling cascades. A given receptor and agonist pair, produce multiple effects upon cellular physiology which can be opposite in different cell types. The production of variable cellular effects via the signaling of the same GPCR in different cell types is a result of signal organization in space and time (compartmentation). This organization is usually based upon the physical and chemical properties of the membranes in which the GPCRs reside and the repertoire of downstream effectors and co-factors that are available at that location. In this review we explore mechanisms of GPCR signal compartmentation and broadly review the state-of-the-art methodologies which can be utilized to study them. We provide a clear rationale for a ‘localized’ approach to the study of the pharmacology and physiology of GPCRs and particularly the secondary messenger cAMP

Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography.

With scanning ion conductance microscopy (SICM), a noncontact scanning probe technique, it is possible both to obtain information about the surface topography of live cells and to apply molecules onto specific nanoscale structures. The technique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surfaces of various cell types. The heart muscle cells, i.e., the cardiomyocytes, possess a highly elaborate, unique surface topography including transverse-tubule (T-tubule) openings leading into a cell internal system that exclusively harbors many proteins necessary for the cell's physiological function. Here, we applied isoproterenol into these surface openings by changing the applied voltage over the SICM nanopipette. To determine the grade of precision of our application we used finite-element simulations to investigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytes using SICM and then determined the electrophoretic mobility of isoproterenol in a high ion solution to be -7 × 10(-9) m(2)/V s. The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove, and especially to the first T-tubule opening, where the concentration is ∼6.5 times higher compared to on a flat surface under the same delivery settings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importance of topography in agonist delivery. In conclusion, SICM, unlike other techniques, can reliably deliver precise quantities of compounds to the T-tubules of cardiomyocytes