Luca Mendler

On Marathons and Sprints: An Integrated Quantitative Proteomics and Transcriptomics Analysis of Differences Between Slow and Fast Muscle Fibers

Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and α-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented resolution.

Regenerating soleus and extensor digitorum longus muscles of the rat show elevated levels of TNF-α and its receptors, TNFR-60 and TNFR-80

We measured the mRNA and protein levels of tumor necrosis factor‐α (TNF‐α) and the transcript levels of its receptors (TNFR‐60 and TNFR‐80) in the rat soleus (slow twitch) and extensor digitorum longus (EDL; fast twitch) muscles regenerating from notexin‐induced necrosis. On the first day after administration of the toxin, when most fibers were necrotic and invaded by inflammatory cells/macrophages, dramatic increases of transcript and protein levels of TNF‐α and of the mRNA levels of its receptors were observed. The transcript levels of TNF‐α and TNFR‐60, but not of TNFR‐80, showed a second but smaller increase at the time when newly formed muscle fibers became reinnervated. In situ hybridization showed that on day 1, during the phase of extensive necrosis, the transcript of TNF‐α was abundantly present and on day 4 of regeneration it was most often seen in areas devoid of desmin. The mRNA level of TNF‐α was not detectable in BC3H1‐ and C2C12‐cultured myoblasts and it was low in freeze‐injured muscle, corresponding to the relatively mild degree of inflammation elicited by freezing. Therefore, our results are most consistent with the view that inflammatory cells/macrophages are the main source of TNF‐α.

Myostatin levels in regenerating rat muscles and in myogenic cell cultures.

Myostatin is a newly described member of the TGF-β superfamily acting as a secreted negative regulator of skeletal muscle mass in several species, but whose mode of action remains largely unknown. In the present work, we followed the myostatin mRNA and protein levels in rat soleus and extensor digitorum longus (EDL) muscles regenerating in vivo from notexin-induced necrosis, and the myostatin transcript levels in two different in vitro myogenic differentiation models: i.e. in mouse BC3H1 and C2C12 cultured cells. The in vivo regenerating rat skeletal muscles showed a characteristic time-dependent expression of myostatin mRNA. After notexin injection, the transcript levels dropped below the detection limit on day 1 in soleus and close to the detection limit on day 3 in EDL, then increased to a maximum on day 7 in soleus and after 28 days finally reached the control values in both types of muscles. In contrast, the myostatin protein levels increased dramatically on the first days of regeneration in both muscles, i.e. at the time when its transcript level was low. Later on the myostatin protein level gradually declined to normal in soleus while in EDL it stayed high some days longer and decreased to normal on days 21–28. In vitro proliferating myoblasts produced low level of myostatin mRNA, which increased upon induction of differentiation suggesting that functional innervation is no prerequisite for myostatin expression. Myostatin production in vitro seems not to be dependent on myocyte fusion either, since it is observed in differentiated BC3H1 cells, which are defective in myofiber formation.

Myostatin and IGF-I signaling in end-stage human heart failure: a qRT-PCR study

BackgroundMyostatin (Mstn) is a key regulator of heart metabolism and cardiomyocyte growth interacting tightly with insulin-like growth factor I (IGF-I) under physiological conditions. The pathological role of Mstn has also been suggested since Mstn protein was shown to be upregulated in the myocardium of end-stage heart failure. However, no data are available about the regulation of gene expression of Mstn and IGF-I in different regions of healthy or pathologic human hearts, although they both might play a crucial role in the pathomechanism of heart failure.MethodsIn the present study, heart samples were collected from left ventricles, septum and right ventricles of control healthy individuals as well as from failing hearts of dilated (DCM) or ischemic cardiomyopathic (ICM) patients. A comprehensive qRT-PCR analysis of Mstn and IGF-I signaling was carried out by measuring expression of Mstn, its receptor Activin receptor IIB (ActRIIB), IGF-I, IGF-I receptor (IGF-IR), and the negative regulator of Mstn miR-208, respectively. Moreover, we combined the measured transcript levels and created complex parameters characterizing either Mstn- or IGF-I signaling in the different regions of healthy or failing hearts.ResultsWe have found that in healthy control hearts, the ratio of Mstn/IGF-I signaling was significantly higher in the left ventricle/septum than in the right ventricle. Moreover, Mstn transcript levels were significantly upregulated in all heart regions of DCM but not ICM patients. However, the ratio of Mstn/IGF-I signaling remained increased in the left ventricle/septum compared to the right ventricle of DCM patients (similarly to the healthy hearts). In contrast, in ICM hearts significant transcript changes were detected mainly in IGF-I signaling. In paralell with these results miR-208 showed mild upregulation in the left ventricle of both DCM and ICM hearts.ConclusionsThis is the first demonstration of a spatial asymmetry in the expression pattern of Mstn/IGF-I in healthy hearts, which is likely to play a role in the different growth regulation of left vs. right ventricle. Moreover, we identified Mstn as a massively regulated gene in DCM but not in ICM as part of possible compensatory mechanisms in the failing heart.

Myostatin Regulates Energy Homeostasis in the Heart and Prevents Heart Failure

Rationale: Myostatin is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes, including enhanced insulin sensitivity. However, the function of myostatin in the heart is barely understood, although it is upregulated in the myocardium under several pathological conditions. Objective: Here, we aimed to decipher the role of myostatin and myostatin-dependent signaling pathways for cardiac function and cardiac metabolism in adult mice. To avoid potential counterregulatory mechanisms occurring in constitutive and germ-line–based myostatin mutants, we generated a mouse model that allows myostatin inactivation in adult cardiomyocytes. Methods and Results: Cardiac MRI revealed that genetic inactivation of myostatin signaling in the adult murine heart caused cardiac hypertrophy and heart failure, partially recapitulating effects of the age-dependent decline of the myostatin paralog growth and differentiation factor 11. We found that myostatin represses AMP-activated kinase activation in the heart via transforming growth factor-&bgr;–activated kinase 1, thereby preventing a metabolic switch toward glycolysis and glycogen accumulation. Furthermore, myostatin stimulated expression of regulator of G-protein signaling 2, a GTPase-activating protein that restricts Gaq and Gas signaling and thereby protects against cardiac failure. Inhibition of AMP-activated kinase in vivo rescued cardiac hypertrophy and prevented enhanced glycolytic flow and glycogen accumulation after inactivation of myostatin in cardiomyocytes. Conclusions: Our results uncover an important role of myostatin in the heart for maintaining cardiac energy homeostasis and preventing cardiac hypertrophy.

The Ubiquitin-Like SUMO System and Heart Function: From Development to Disease

SUMOylation is a ubiquitin-related transient posttranslational modification pathway catalyzing the conjugation of small ubiquitin-like modifier (SUMO) proteins (SUMO1, SUMO2, and SUMO3) to lysine residues of proteins. SUMOylation targets a wide variety of cellular regulators and thereby affects a multitude of different cellular processes. SUMO/sentrin-specific proteases are able to remove SUMOs from targets, contributing to a tight control of SUMOylated proteins. Genetic and cell biological experiments indicate a critical role of balanced SUMOylation/deSUMOylation for proper cardiac development, metabolism, and stress adaptation. Here, we review the current knowledge about SUMOylation/deSUMOylation in the heart and provide an integrated picture of cardiac functions of the SUMO system under physiologic or pathologic conditions. We also describe potential therapeutic approaches targeting the SUMO machinery to combat heart disease.

Regeneration of Reinnervated Rat Soleus Muscle Is Accompanied by Fiber Transition Toward a Faster Phenotype

The functional recovery of skeletal muscles after peripheral nerve transection and microsurgical repair is generally incomplete. Several reinnervation abnormalities have been described even after nerve reconstruction surgery. Less is known, however, about the regenerative capacity of reinnervated muscles. Previously, we detected remarkable morphological and motor endplate alterations after inducing muscle necrosis and subsequent regeneration in the reinnervated rat soleus muscle. In the present study, we comparatively analyzed the morphometric properties of different fiber populations, as well as the expression pattern of myosin heavy chain isoforms at both immunohistochemical and mRNA levels in reinnervated versus reinnervated-regenerated muscles. A dramatic slow-to-fast fiber type transition was found in reinnervated soleus, and a further change toward the fast phenotype was observed in reinnervated-regenerated muscles. These findings suggest that the (fast) pattern of reinnervation plays a dominant role in the specification of fiber phenotype during regeneration, which can contribute to the long-lasting functional impairment of the reinnervated muscle. Moreover, because the fast II fibers (and selectively, a certain population of the fast IIB fibers) showed better recovery than did the slow type I fibers, the faster phenotype of the reinnervated-regenerated muscle seems to be actively maintained by selective yet undefined cues.

Expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases in the rat extensor digitorum longus (EDL) muscle regenerating from notexin-induced necrosis.

The level of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) mRNAs and proteins have been assessed by␣RT-PCR,␣immunoblotting and immunocytochemistry in the rat extensor digitorum longus (EDL) muscles during regeneration from notexin-induced necrosis. As a result of the necrosis, SERCA1 and SERCA2 declined on days 1 and 3 after␣administration of the toxin. Thereupon the mRNA of the fast isoform SERCA1 rapidly increased between days 5 and 10 to the normal level. The mRNA level of the “housekeeping” SERCA2b isoform increased markedly during the actual necrosis at days 1 and 5, probably due to invading cells. Then the mRNA level of the neonatal SERCA1b splice variant increased first, and exceeded the level of the adult SERCA1a transcript on day 5. At later stages of regeneration the neonatal form was gradually replaced by the adult SERCA1a form, thus recapitulating similar changes known to occur during normal ontogenesis. Along with S ERCA1, the levels of the slow isoform (SERCA2a) mRNA and protein increased on day 5, but the␣SERCA2a mRNA levels never rose above 10% of SERCA1 and after 10 days gradually declined again. In the normal and␣regenerated muscles, SERCA1 was expressed in 97% of the fibres which accounted for the population of fast-twitch fibres␣(type IIa, type IIb and probably type IIx/d). SERCA2a was present in 6% of the fibres of normal muscle (mostly in the␣slow-twitch type I fibres). At the end of regeneration the number of fibres expressing SERCA2a was twice as high and␣were␣found in small groups, unlike in normal EDL, but about 50% of these clustered fibres also expressed SERCA1.

Myostatin induces interstitial fibrosis in the heart via TAK1 and p38

Myostatin, a member of the TGF-β superfamily of secreted growth factors, is a negative regulator of skeletal muscle growth. In the heart, it is expressed at lower levels compared to skeletal muscle but up-regulated under disease conditions. Cre recombinase-mediated inactivation of myostatin in adult cardiomyocytes leads to heart failure and increased mortality but cardiac function of surviving mice is restored after several weeks probably due to compensatory expression in non-cardiomyocytes. To study long-term effects of increased myostatin expression in the heart and to analyze the putative crosstalk between cardiomyocytes and fibroblasts, we overexpressed myostatin in cardiomyocytes. Increased expression of myostatin in heart muscle cells caused interstitial fibrosis via activation of the TAK-1-MKK3/6-p38 signaling pathway, compromising cardiac function in older mice. Our results uncover a novel role of myostatin in the heart and highlight the necessity for tight regulation of myostatin to maintain normal heart function.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

ABSTRACT Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-&bgr;1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2−/− relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2−/− fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.