“Turnover Proteome” of Human Atrial Trabeculae

Abstract

Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.

Mitochondrial and Contractile Function of Human Right Atrial Tissue in Response to Remote Ischemic Conditioning

“…Right atrial trabeculae were electrically paced with rectangular pulses (5‐ms) field stimulation at 1 Hz and then gradually prestretched to their maximal force of contraction, as previously described 30, 31. Maximal force of contraction was reached when further stretching did not cause greater force of contraction 31.…”

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

“…Inserted tubes enable gas supply and buffer changes. After pacing atrial trabeculae with rectangular pulses (5 ms) and a field stimulation at 1 Hz, trabeculae are gradually pre-stretched to their maximal force of contraction [ 267 , 311 ]. When further stretching does not cause greater force of contraction [ 311 ], stretch is reduced to 90% of the length with such maximal force [ 99 ].…”

Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

“…The SILAC strategy with 13 C-labeled amino acids has been widely used to measure protein turnover rates (5,7,10). However, this strategy is not well suited to fully labeling plant cells for multiple reasons.…”

“…Some are direct measurements of endogenous proteins using isotope labeling methods including both radioactive and stable isotope labeling (5,(7)(8)(9)(10), whereas others use stable or transient transgenic techniques and a range of tags and markers (11,12). The clearest advantage of isotope labeling approaches is that the tags are very subtle with little or no impact on cellular processes and allow the fully functional proteins being assessed to be produced and distributed within cells in a normal context.…”