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2. Preparing Excitable Cardiac Papillary Muscle and Cardiac Slices for Functional Analyses

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   Palmer, Bradley M.; Bell, Stephen P. (March 2022, Frontiers in Physiology)

   While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing cardiac papillary muscle and cardiac slices, which represent macroscopic experimental model systems with fully intact intercellular and intracellular structures. The maintenance of these in vivo structures for experimentation in vitro have made these model systems especially useful for testing the functional effects of protein mutations and pharmaceutical candidates. We provide solutions recipes for dissection and recording, instructions for removing and preparing the cardiac papillary muscles, as well as instruction for preparing cardiac slices. These instructions are suitable for beginning experimentalists but may be useful for veteran muscle physiologists hoping to reacquaint themselves with macroscopic functional analyses. 

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   Masanori Aikawa (Ed.)

   As binary switches, RAS proteins switch to an ON/OFF state during signaling and are on a leash under normal conditions. However, in RAS-related diseases such as cancer and RASopathies, mutations in the genes that regulate RAS signaling or the RAS itself permanently activate the RAS protein. The structural basis of this switch is well understood; however, the exact mechanisms by which RAS proteins are regulated are less clear. RAS/MAPK syndromes are multisystem developmental disorders caused by germline mutations in genes associated with the RAS/mitogen-activated protein kinase pathway, impacting 1 in 1,000–2,500 children. These include a variety of disorders such as Noonan syndrome (NS) and NS-related disorders (NSRD), such as cardio facio cutaneous (CFC) syndrome, Costello syndrome (CS), and NS with multiple lentigines (NSML, also known as LEOPARD syndrome). A frequent manifestation of cardiomyopathy (CM) and hypertrophic cardiomyopathy associated with RASopathies suggest that RASopathies could be a potential causative factor for CM. However, the current supporting evidence is sporadic and unclear. RASopathy-patients also display a broad spectrum of congenital heart disease (CHD). More than 15 genes encode components of the RAS/MAPK signaling pathway that are essential for the cell cycle and play regulatory roles in proliferation, differentiation, growth, and metabolism. These genes are linked to the molecular genetic pathogenesis of these syndromes. However, genetic heterogeneity for a given syndrome on the one hand and alleles for multiple syndromes on the other make classification difficult in diagnosing RAS/MAPK-related diseases. Although there is some genetic homogeneity in most RASopathies, several RASopathies are allelic diseases. This allelism points to the role of critical signaling nodes and sheds light on the overlap between these related syndromes. Even though considerable progress has been made in understanding the pathophysiology of RASopathy with the identification of causal mutations and the functional analysis of their pathophysiological consequences, there are still unidentified causal genes for many patients diagnosed with RASopathies. 

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