"Turnover proteome" of human atrial trabeculae.
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.
Lampert
Florian M
FM
Department of Research, University Hospital Basel, Hebelstrasse 20, CH 4031 Basel, Switzerland.
Matt
Peter
P
Grapow
Martin
M
Lefkovits
Ivan
I
Zerkowski
Hans-Reinhard
HR
Grussenmeyer
Thomas
T
D000199:Actins / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D006321:Heart / Q000502:physiology; D006325:Heart Atria / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D009119:Muscle Contraction; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D016622:Silver Staining
D000199:Actins; D010455:Peptides; D020543:Proteome