Morano Lab Header

Morano Lab

Molecular Muscle Physiology


Our objective is to characterize and to understand physiological features of cardiac, skeletal, and smooth muscle of male and female individuals.

We perform that on the transcriptional, translational, and functional levels, using molecular biology, biochemical, cell-biological, and biophysical methods. Basic characterization of muscle functions of both sexes will help us to understand the distinct manifestations of muscle diseases.This is fundamental in order to develop new therapeutic strategies to treat muscle diseases, e.g. hypertrophic and dilative cardiomyopathies.

Therefore, our lab investigates the role of estrogen receptors (ER) on gene expression regulation in cardiomyocytes, in particular on the identification and functions of new co-regulators of the estrogen-activated ER, which modify the transcriptional activities of ER. To understand the energy balancing mechanisms of muscle, we are investigating the biogenesis, structure, and function of mitochondria in muscle.

Furthermore, we are studying the multitude functions of the key protein of Ca2+ activation of muscle, namely the voltage-dependent Ca2+ channel together with an interacting protein partner, the 700 kDa scaffolding protein Ahnak. The molecular mechanisms of depression of Ca2+ activation and, hence contractility of cardiomyocytes by the fatty acid binding proteins FABP3 and FABP4 are part of our ongoing research activities. Type II myosin and its associated light chains are the molecular motors of muscle contraction. Using transgenic animals we are investigating the role of normal essential myosin light chains (ELC) and mutated ELC associated with cardiomyopathy on cardiomyocyte contraction.



Contracting adult rat cardiomyocyte electrically stimulated at 1Hz

Contraction of all muscle types is elicited by increasing myoplasmic Ca2+ and interaction of Type II myosins with thin (actin) filaments. In striated muscle, Ca2+ bind to troponin C, which turn the thin filament “on”, allowing myosin force-generating actin interactions. In smooth muscle cells, Ca2+ bind to calmodulin which then activate myosin light chain kinase. Phosphorylation of the 20kDa light chain of smooth muscle myosins then allows forcegenerating actin interaction. We are studying the functional roles of subunits of key proteins of Ca2+ handling and force generation, i.e. the L-type Ca2+ channel and type II myosins in striated and smooth muscle. Any change of these proteins by mutation, differential gene expression, alternative splicing of the transcripts, or post-translational modification modulate striated and smooth muscle function. Understanding muscle contraction regulation at the molecular and functional levels provide the opportunity to develop new therapeutic strategies for the treatment of cardiovascular and skeletal muscle dysfunction.

Current Projects

Essential myosin light chain (ELC) functions in the heart


In the adult human heart two ELC isoforms are expressed, namely an atrial-specific (MYL4, ALC-1, accession NP_001002841) and a ventricular-specific (MYL3, VLC-1, accession NP_000249) isoform. ELCs bind with their N-terminus to actin and with their C-Terminus to IQ1 of the myosin lever arm (Figure 1). ALC-1 is preferentially targeted into sarcomeres of human and rodent cardiomyocytes. Most patients with hypertrophic cardiomyopathy and congenital heart diseases re-express hALC-1 in their ventricles, partially replacing the VLC-1 isoform. The VLC-1-to-ALC-1 isoform shift induced a pronounced positive inotropic effect.

The molecular basis for the isoform-specific sarcomeric sorting pattern and the molecular mechanisms of ALC-1 inotropy are not yet understood. In this project we test the hypothesis that different binding affinities of the C-terminus of essential myosin light chain (ELC) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity.

Hypertrophic Cardiomyopathy associates with five mutations in the essential ventricular myosin light chain gene (MYL3, AC_000135) (M149V, E143K, A57G, E56G, R154H).The pathomechanism of MYL3 mutations, however, is not yet understood. In this project, we will investigate the functional consequences of MYL3 mutations. We employ analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of recombinant head-rod fragments of cardiac β-myosin heavy chain and ELC isoforms. Cellular functions of ELC isoforms will be investigated by monitoring shortening and intracellular free Ca2+ (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using ELC isoforms or β-galactosidase as expression cassettes. We will generate transgenic mouse lines overexpressing normal or mutated ELC isoforms. In addition, we elaborate a structural model which explains the cis-inhibitory action of the IQ2 domain on myosin function.

Figure 1: 3D-model of the actomyosin complex.
(A) Gauss–Connolly surfaces are used to visualize the molecular complex. Actin units are coloured orange, brown, and yellow. The myosin S1 head (green), the regulatory light chain (red), the shortened essential light chain (white), the 46 N-terminal residues of A1 (blue), and clusters of acidic residues on actin (pink) are shown. (B) More detailed view on the potential interaction of N-terminal APKK of A1 with acidic residues on actin. Ionic interactions between lysine residues (K3 and K4) of APKK and acidic residues (E361 and E364) on actin were assumed.


The role of myomesin missense mutations on the genesis of hypertrophic cardiomyopathy


Hypertrophic cardiomyopathy (HCM) is a common cause of sudden cardiac death in young people. Three missense mutations in the myomesin gene have recently been detected in patients with HCM. These mutations are located close to the myosin and titin binding sites and the dimerization region. The aim of the project is to study the molecular pathomechanisms causing the development of HCM by myomesin mutations (e.g. V1490I). Therefore, functional properties of mutated recombinant fusion proteins will be analyzed, i.e. force measurements of poly-myomesin by atomic force microscopy, structural properties by circular dichroism and melting curves, and cellular functions by transient expression of normal and mutated myomesin domains in cardiomyoblasts. Transgenetic rat lines which overexpress functionally relevant myomesin mutations will be generated in order to investigate their potential to cause HCM.

Ahnak1 – a novel, prominent modulator of cardiac L-type Ca2+channels


Ahnak1 is located at the sarcolemma and T-tubuli of cardiomyocytes indirectly associated with the voltagedependent L-type Ca2+ channel (L-VDCC) via its β2-subunit. The goal of this study will be understanding the interactions of ahnak1 with L-VDCC, i.e. α1C and β2, and their modulation by β-adrenergic stimulation, mutations, and PKC activation (Figure 2).This will be achieved by heterologous expression of ahnak1 fragments in Xenopus oocytes and HEK cells and the characterization of their effects on cardiac and smooth muscle LVDCC. Furthermore, reconstitution of β-adrenergic modulation of L-VDCC in heterologous expression systems, and elucidation of the role of ahnak1, Cavβ and other signaling components in PKA and PKC modulation will be studied.

Figure 2: Proposed model for sympathetic control of ICaL by ahnak1.
Under basal conditions, ICaL carried by the α1C-subunit is repressed by strong ahnak1/β2-subunit binding (left panel). Upon sympathetic stimulation, PKA sites in ahnak1 and/or in β2 become phosphorylated. This releases the β2-subunit from ahnak1 inhibition resulting in increased ICaL.


The functional role of the ahnak protein family in adult skeletal muscle fibers


The aim of the project is the elucidation of the role of ahnak protein family in skeletal muscle fibers. The transmembrane protein dysferlin seems to anchor ahnak1 and ahnak2 to the sarcolemma, thus providing a membrane-stabilizing dysferlin-ahnak-actin complex. We investigate whether the ahnak protein family is important for membrane stability and Ca2+ handling of skeletal muscle fibers. A laser-assisted membrane resealing (Figure 3) and a Fura2-based fluorescence assay of electrically stimulated enzymatically isolated single skeletal muscle fibers from mouse Flexor digitorum brevis will be applied.

Figure 3: Membrane resealing assay performed on wild-type mouse single skeletal muscle fibres.
Membrane damage (5x5μm) was induced with a two-photon confocal laser-scanning microscope (LSM 510 META, Zeiss) coupled to a 488-nm Argon Ti: Sapphire Laser in the presence of FM 1-43FX fluorescence dye. Top: Plot of fluorescence intensity (n=10) against time in the presence (red line) and absence (black line) of Ca2+. Data are means +/-SEM. Bottom: fluorescence obtained in the presence (right) and absence (left) of Ca2+.


Identification of new adipocyte-derived cardiodepressant factors


Recently, we showed that cultivated human adipocyte secrete factors which depress contraction of cardiomyocytes (Figure 4). The aim of this project is to identify adipocyte-derived cardiodepressant factors.

Figure 4: Effect of adipocyte-conditioned medium (AM) on shortening amplitude (top trace) and Fura- 2 signal (bottom trace) of an adult rat cardiomyocyte.
A, B) Representative chart recordings of shortening amplitudes.

We will assess the role of different adipose tissue depots in producing these cardiodepressant factors in our in vitro adipocyte/cardiomyocyte and adipocyte/isolated perfused heart models (Figure 5). Finally, the adipocyte-derived cardiodepressant factors will be analysed in vivo in microdialysis samples of subcutaneous adipose tissue of obese patients as well as in serum of our obese patient cohort. The resulting data will allow the development of risk profiling and novel therapeutic strategies for heart dysfunction in obese patients.

Figure 5:
Electrically stimulated isolated perfused rat heart (Langendorff).



Prof. Dr. Ingo Morano
Prof. Dr. Ingo Morano
Group Leader
Max-Delbrück-Centrum für Molekulare Medizin (MDC)
Robert-Rössle-Str. 10
13092 Berlin, Germany
31.1: Max Delbrück House
Room 0114.1
(030) 9406 2313
Manuela Kaada
(030) 9406 2209
31.1: Max Delbrück House
Room 0116