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Technical Challenges

Unique aspects of the cardiomyocytes lead to additional technological challenges. Technological advancements in mass spectrometers and separation technologies have generally increased the number of proteins identified in a proteomics discovery paper by ~5 fold since 2002.  However, this achievement has not been observed in cardiac muscle. Reasons for this are multi-factorial, however, chief among them is the fact that cardiac muscle has a large dynamic range due to dominance of the myofilament and mitochondrial subproteomes, and this generally requires extensive fractionation or targeted enrichment (4, 9-11) to observe low abundance proteins.  This unique aspect of cardiac muscle is due to its intrinsic physiology – the sustained intense energetic requirements for continuous contractile activity and need for minute-to-minute adaptation of heart rate, blood pressure and cardiac output.

The challenge of cardiac protein isoforms.  Another unique aspect of the myocyte proteome is the large number of cardiac-specific isoforms and they can be regulated by different PTMs.  For example, the cardiac isoform of troponin I (cTnI) regulates contraction/relaxation but unlike the skeletal muscle isoforms is regulated by protein kinase A (PKA) phosphorylation. It can also undergo O-GlcNAcylation and be specifically degraded under different pathological conditions (12-17). A small degree of change to these PTMs can result in large physiological alterations (e.g. 15 percent substitution of the truncated form results in ~50% reduced contractility (15)). In vivo human hearts only 2 of the 6 potential phosphorylated sites on cTnI are modified (18) and, according to data from animal models this would have a substantial effects on responses to tachycardia and afterload (19, 20). Since subtle PTM changes can have large functional consequences special tools must be developed for cardiovascular proteomics. Tools that are able to i) assess multiple PTMs, ii) quantify each modified residue and iii) be accurate so that the low abundance of these can be quantified.

The challenge of terminal differentiation. In addition to the problems outlined above, the final issue for cardiac muscle proteomic studies is that there are no dividing cell culture systems for ventricular cardiomyocytes so that approaches dependent upon complete saturation metabolic labeling, like SILAC, are not possible. Therefore, unique metabolic labeling strategies or alternative technologies will need to be extended or adapted to detail cardiac protein changes, as described below.