Research Report 2010 - MDC

Ingo L. MoranoStructure of the GroupGroup LeaderProf. Dr. Ingo MoranoScientistsDr. Hannelore HaaseDr. Daria PetzholdDr. Andreas MargGraduate StudentsChristiane LookRomy SiegertJanine LossieInes PankonienLena MartinMaria BöhmerMolecular Muscle PhysiologyContraction of all muscle types is elicited by increasing myoplasmic Ca 2+ and interaction ofType II myosins with thin (actin) filaments. In striated muscle, Ca 2+ bind to troponin C,which turn the thin filament “on”, allowing myosin force-generating actin interactions. Insmooth muscle cells, Ca 2+ bind to calmodulin which then activate myosin light chain kinase.Phosphorylation of the 20kDa light chain of smooth muscle myosins then allows forcegeneratingactin interaction. We are studying the functional roles of subunits of key proteinsof Ca 2+ handling and force generation, i.e. the L-type Ca 2+ channel and type II myosins instriated and smooth muscle. Any change of these proteins by mutation, differential geneexpression, alternative splicing of the transcripts, or post-translational modification modulatestriated and smooth muscle function. Understanding muscle contraction regulation at themolecular and functional levels provide the opportunity to develop new therapeutic strategiesfor the treatment of cardiovascular and skeletal muscle dysfunction.Essential myosin light chain (ELC) functions in theheartDaria Petzhold, Janine Lossie, Maria Böhmer, BurcuSimsek; Ralf Meißner, Petra Sakel, Saskia ReichertIn the adult human heart two ELC isoforms areexpressed, namely an atrial-specific (MYL4, ALC-1, accessionNP_001002841) and a ventricular-specific (MYL3,VLC-1, accession NP_000249) isoform. ELCs bind withtheir N-terminus to actin and with their C-Terminus toIQ1 of the myosin lever arm (Figure 1). ALC-1 is preferentiallytargeted into sarcomeres of human and rodentcardiomyocytes. Most patients with hypertrophic cardiomyopathyand congenital heart diseases re-expresshALC-1 in their ventricles, partially replacing the VLC-1isoform. The VLC-1-to-ALC-1 isoform shift induced a pronouncedpositive inotropic effect.The molecular basis for the isoform-specific sarcomericsorting pattern and the molecular mechanisms of ALC-1 inotropy are not yet understood. In this project we testthe hypothesis that different binding affinities of the C-terminus of essential myosin light chain (ELC) isoformsto the IQ1 motif of the myosin lever arm provide amolecular basis for distinct sarcomeric sorting andinotropic activity.Hypertrophic Cardiomyopathy associates with fivemutations in the essential ventricular myosin lightchain gene (MYL3, AC_000135) (M149V, E143K, A57G,E56G, R154H). The pathomechanism of MYL3 mutations,however, is not yet understood. In this project, we willinvestigate the functional consequences of MYL3 mutations.We employ analytical ultracentrifugation, circulardichroism, and surface plasmon resonance spectroscopyto investigate structural properties, secondarystructures, and protein-protein interactions of recombinanthead-rod fragments of cardiac β-myosin heavychain and ELC isoforms. Cellular functions of ELC isoformswill be investigated by monitoring shorteningand intracellular free Ca 2+ (Fura-2) of adult rat cardiomyocytesinfected with adenoviral (Ad) vectors using ELCisoforms or β-galactosidase as expression cassettes. Wewill generate transgenic mouse lines overexpressingnormal or mutated ELC isoforms. In addition, we elaboratea structural model which explains the cis-inhibitoryaction of the IQ2 domain on myosin function.12 Cardiovascular and Metabolic Disease Research