Peterskelley3387

Interventional cardiology has witnessed tremendous changes over the years from a mainly diagnostic approach in an elective population to therapeutic strategies in critically ill patients. Currently, we can treat a broad spectrum of coronary artery, peripheral artery, and structural heart diseases with less invasive, percutaneous approaches that we did not anticipate to be possible just a decade ago. It is certain that the interventional techniques will see further development and we will be able to treat by percutaneous methods more conditions previously thought beyond our reach. Regardless of the advances in catheter-based diagnostic and therapeutic techniques, one thing remains constant. They all require vascular access. And, vascular access is the first technical part of any percutaneous cardiovascular procedure that can determine its overall success. High-quality data together with the availability of training courses for interventional cardiologists and fellows-in-training ensure systematic use of the transradial approach (TRA) which has demonstrated a considerable benefit compared to transfemoral approach both in chronic and acute coronary syndromes. Constant improvement of TRA techniques will further facilitate transradial endovascular and structural interventions, and the growing use for high-risk and complex percutaneous coronary interventions. A continuously growing body of evidence is focused on surpassing current TRA limitations (specifically radial artery occlusion) and expanding alternative vascular accesses such as transulnar approach or distal TRA ("snuff-box" technique). Should this downsizing trend continue, we could see a further paradigm shift toward using the snuff-box technique.Voltage sensing phosphatase (VSP), consists of a voltage sensor domain (VSD) like that found in voltage-gated ion channels and a phosphoinositide (PIP) phosphatase region exhibiting remarkable structural similarity to a tumor suppressor enzyme, PTEN. Membrane depolarization activates the enzyme activity through tight coupling between the VSD and enzyme region. The VSD of VSP has a unique nature; it is a self-contained module that can be transferred to other proteins, conferring voltage sensitivity. Thanks to this nature, numerous versions of gene-encoded voltage indicators (GEVIs) have been developed through combination of a fluorescent protein with the VSD of VSP. In addition, VSP itself can also serve as a tool to alter PIP levels in cells. selleck compound Cellular levels of PIPs, PI(4,5)P2 in particular, can be acutely and transiently reduced using a simple voltage protocol after heterologous expression of VSP. Recent progress in our understanding of the molecular structure and mechanisms underlying VSP facilitates optimization of its molecular properties for its use as a molecular tool.Fluorescence spectroscopy and microscopy are non-destructive methods that provide real-time measurements of ion channel structural dynamics. As such, they constitute a direct path linking the high-resolution structural models from X-ray crystallography and cryo-electron microscopy with the high-resolution functional data from ionic current measurements. The utility of fluorescence as a reporter of channel structure is limited by the palette of available fluorophores. Thiol-reactive fluorophores are small and bright, but are restricted in terms of the positions on a protein that can be labeled and present significant issues with background incorporation. Genetically encoded fluorescent protein tags are specific to a protein of interest, but are very large and usually only used to label the free N- and C-termini of proteins. L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP) is a fluorescent amino acid that can be specifically incorporated into virtually any site on a protein of interest using amber stop-codon suppression. Due to its environmental sensitivity and potential as a donor in fluorescence resonance energy transfer experiments, it has been adopted by numerous investigators to study voltage, ligand, and temperature-dependent activation of a host of ion channels. Simultaneous measurements of ionic currents and ANAP fluorescence yield exceptional mechanistic insights into channel function. In this chapter, I will summarize the current literature regarding ANAP and ion channels and discuss the practical aspects of using ANAP, including potential pitfalls and confounds.Sudden cardiac death continues to have a devastating impact on public health prompting the continued efforts to develop more effective therapies for cardiac arrhythmias. Among different approaches to normalize function of ion channels and prevent arrhythmogenic remodeling of tissue substrate, cardiac cell and gene therapies are emerging as promising strategies to restore and maintain normal heart rhythm. Specifically, the ability to genetically enhance electrical excitability of diseased hearts through voltage-gated sodium channel (VGSC) gene transfer could improve velocity of action potential conduction and act to stop reentrant circuits underlying sustained arrhythmias. For this purpose, prokaryotic VGSC genes are promising therapeutic candidates due to their small size ( less then 1kb) and potential to be effectively packaged in adeno-associated viral (AAV) vectors and delivered to cardiomyocytes for stable, long-term expression. This article describes a versatile method to discover and characterize novel prokaryotic ion channels for use in gene and cell therapies for heart disease including cardiac arrhythmias. Detailed protocols are provided for (1) identification of potential ion channel candidates from large genomic databases, (2) candidate screening and characterization using site-directed mutagenesis and engineered human excitable cell system and, (3) candidate validation using electrophysiological techniques and an in vitro model of impaired cardiac impulse conduction.Patch clamp recording enabled a revolution in cellular electrophysiology, and is useful for evaluating the functional consequences of ion channel gene mutations or variants associated with human disorders called channelopathies. However, due to massive growth of genetic testing in medical practice and research, the number of known ion channel variants has exploded into the thousands. Fortunately, automated methods for performing patch clamp recording have emerged as important tools to address the explosion in ion channel variants. In this chapter, we present our approach to harnessing automated electrophysiology to study a human voltage-gated potassium channel gene (KCNQ1), which harbors hundreds of mutations associated with genetic disorders of heart rhythm including the congenital long-QT syndrome. We include protocols for performing high efficiency electroporation of heterologous cells with recombinant KCNQ1 plasmid DNA and for automated planar patch recording including data analysis. These methods can be adapted for studying other voltage-gated ion channels.