Fielitz Lab

Skeletal muscle wasting with accompanying cachexia is a life threatening complication in congestive heart failure. The molecular mechanisms are imperfectly understood, although an activated renin–angiotensin aldosterone system has been implicated. Angiotensin (Ang) II induces skeletal muscle atrophy in part by increased muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1/Trim63) expression, which may involve protein kinase D1 (PKD1). In a recent study we elucidated the molecular mechanism of Ang II–induced skeletal muscle wasting. In a cDNA expression screen we identified the lysosomal hydrolase-coordinating transcription factor EB (TFEB) as novel regulator of the human MuRF1/Trim63 promoter. TFEB played a key role in regulating Ang II–induced skeletal muscle atrophy by transcriptional control of MuRF1/Trim63 via conserved E-box elements. Inhibiting TFEB with small interfering RNA prevented Ang II–induced MuRF1/Trim63 expression and muscle atrophy. The histone deacetylase-5 (HDAC5), which was directly bound to and colocalized with TFEB, inhibited TFEB-induced MuRF1/Trim63 expression. The inhibition of TFEB by HDAC5 was reversed by PKD1, which was associated with HDAC5 and mediated its nuclear export. Mice lacking PKD1 in skeletal myocytes were resistant to Ang II–induced muscle wasting. In our study we proposed that elevated Ang II serum concentrations, as occur in patients with congestive heart failure, could activate the PKD1/HDAC5/TFEB/MuRF1 pathway to induce skeletal muscle wasting. Now we are working on further molecular mechanisms involved in this signalling pathway..

Striated muscle structure and function is maintained by precise control of protein synthesis and degradation; abnormalities in these processes can give rise to myopathies. The UPS degrades misfolded proteins. UPS substrate specificity is mediated by E3 ligases, such as RING-finger proteins. Muscle RING-finger (MuRF) proteins 1, 2, and 3 comprise a subfamily of the RING-finger E3 ubiquitin ligases that are specifically expressed in the heart and skeletal muscle. We generated MuRF3 knockout mice and demonstrated that MuRF3 is involved in maintaining cardiac structure and function, and the integrity of the ventricular wall following acute myocardial infarction. Additionally, we found that MuRF1–/–/MuRF3–/– double mutant (DKO) mice display a distinct cardiac and skeletal muscle protein aggregate myopathy. Cardiac and skeletal muscles of DKO mice displayed a striking subsarcolemmal accumulation of myosin (MyHC) accompanied by cardiac hypertrophy, decreased cardiac function and reduced maximal force development of the skeletal muscle (Figure 1). MuRF1 and MuRF3 interact specifically with ?/slow MyHC and MyHCIIa and utilize UbcH5a, -b, and -c as E2 ubiquitin–conjugating enzymes to catalyze the ubiquitinylation and degradation of these proteins. In addition, we generated and phenotypically characterized MuRF2–/–/MuRF3–/– DKO mice. MuRF2–/–/MuRF3–/– DKO mice also showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO skeletal muscle. In addition, a fibre type shift towards slow/type I fibres occurred in MuRF2–/–/MuRF3–/– DKO soleus and extensor digitorum longus. MuRF2–/–/MuRF3–/– DKO hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the MyHC isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in MuRF2–/–/MuRF3–/– DKO hearts. Our data show that all members of the MuRF family have partially redundant functions which are important for maintenance of skeletal muscle and cardiac structure and function in vivo. We are now working on the regulation of function and activity of MuRF proteins.

Clinical data suggested that sepsis and systemic inflammation are major risk factors for ICUAW and CIM and increased serum levels of inflammatory cytokines might induce muscle atrophy in critically ill patients. We reported that the inflammatory cytokine interleukin-6 (IL6) is continuously increased in muscle during inflammation and associated with muscle failure. Increased IL6 expression in muscles of critically ill patients was indicative for inflammation directly occurring in muscle. We reported that inflammation causes acute-phase response (APR) directly in skeletal muscle of critically ill patients. The APR protein serum amyloid A (SAA) 1 was increased in the skeletal muscle of CIM patients were it accumulated in the interstitium, around myofibers. Increased SAA1 expression was inversely correlated with excitability of muscle membranes indicating that SAA1 plays a role in muscle function. We showed that differentiated human and murine myocytes synthesize SAA1, and that SAA1 synthesis was increased by inflammatory cytokines (i.e. IL6, TNF?). Importantly, both IL6 and SAA1 induce atrophy by increasing protein degradation in muscle of patients and rodents. Together, our data indicated that inflammation induces APR directly in muscle possibly contributing to ICUAW. However, the mechanisms how inflammatory cytokines and APR proteins mediate muscle atrophy, and the receptors and signaling pathways involved in this process are unknown.

Figure 1. We show the effects of cardiomyocyte specific PKD1 elimination. After angiotensin II (Ang II) treatment, PKD1 mutant mice (cKO) displayed less cardiac hypertrophy and a reduction in perivascular and interstitial fibrosis (hematoxylin and eosin and Masson's trichrome stainings; top panel). Atrial natriuretic factor (ANF), ß-myosin heavy chain (β-MHC), and procollagen, type I, α2 (Col1α2) expression was also compromised in PKD1 mutant mice (cKO) (bottom panel).