Activation of Tripartite Motif Containing 63 Expression by Transcription Factor EB and Transcription Factor Binding to Immunoglobulin Heavy Chain Enhancer 3 Is Regulated by Protein Kinase D and Class IIa Histone Deacetylases

Rationale: The ubiquitin-proteasome system (UPS) is responsible for skeletal muscle atrophy. We showed earlier that the transcription factor EB (TFEB) plays a role by increasing E3 ubiquitin ligase muscle really interesting new gene-finger 1(MuRF1)/tripartite motif-containing 63 (TRIM63) expression. MuRF 1 ubiquitinates structural proteins and mediates their UPS-dependent degradation. We now investigated how TFEB-mediated TRIM63 expression is regulated. Objective: Because protein kinase D1 (PKD1), histone deacetylase 5 (HDAC5), and TFEB belong to respective families with close structural, regulatory, and functional properties, we hypothesized that these families comprise a network regulating TRIM63 expression. Methods and Results: We found that TFEB and transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) activate TRIM63 expression. The class IIa HDACs HDAC4, HDAC5, and HDAC7 inhibited this activity. Furthermore, we could map the HDAC5 and TFE3 physical interaction. PKD1, PKD2, and PKD3 reversed the inhibitory effect of all tested class IIa HDACs toward TFEB and TFE3. PKD1 mediated nuclear export of all HDACs and lifted TFEB and TFE3 repression. We also mapped the PKD2 and HDAC5 interaction. We found that the inhibitory effect of PKD1 and PKD2 toward HDAC4, HDAC5, and HDAC7 was mediated by their phosphorylation and 14-3-3 mediated nuclear export. Conclusion: TFEB and TFE3 activate TRIM63 expression. Both transcription factors are controlled by HDAC4, HDAC5, HDAC7, and all PKD-family members. We propose that the multilevel PKD/HDAC/TFEB/TFE3 network tightly controls TRIM63 expression

Skeletal Muscle 11beta-HSD1 Controls Glucocorticoid-Induced Proteolysis and Expression of E3 Ubiquitin Ligases Atrogin-1 and MuRF-1

Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx)) and MuRF-1 (Muscle RING-Finger-1). We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [3H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [3H]-cortisone to [3H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12) as well as in primary human myotubes. 11beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p<0.01), suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p<0.001), which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p<0.01, respectively). Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx) completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1

Angiotensin-(1-7) Receptor Mas in Hemodynamic and Thermoregulatory Dysfunction After High-Level Spinal Cord Injury in Mice: A Pilot Study

Spinal cord injury (SCI) above mid-thoracic levels leads to autonomic dysfunction affecting both the cardiovascular system and thermoregulation. The renin-angiotensin system (RAS) which is a potent regulator of blood pressure, including its novel beneficial arm with the receptor Mas could be an interesting target in post-SCI hemodynamics. To test the hypothesis that hemodynamics, activity and diurnal patterns of those are more affected in the Mas deficient mice post-SCI we used a mouse model of SCI with complete transection of spinal cord at thoracic level 4 (T4-Tx) and performed telemetric monitoring of blood pressure (BP) and heart rate (HR). Our data revealed that hypothermia deteriorated physiological BP and HR control. Preserving normothermia by keeping mice at 30°C prevented severe hypotension and bradycardia post-SCI. Moreover, it facilitated rapid return of diurnal regulation of BP, HR and activity in wild type (WT) mice. In contrast, although Mas deficient mice had comparable reacquisition of diurnal HR rhythm, they showed delayed recovery of diurnal rhythmicity in BP and significantly lower nocturnal activity. Exposing mice with T4-Tx (kept in temperature-controlled cages) to 23°C room temperature for one hour at different time-points post-SCI, demonstrated their inability to maintain core body temperature, Mas deficient mice being significantly more impaired than WT littermates. We conclude that Mas deficient mice were more resistant to acute hypotension, delayed nocturnal recovery, lower activity and more severely impaired thermoregulation. The ambient temperature had significant effect on hemodynamics and, thus it should be taken into account when assessing cardiovascular parameters post-SCI in mice

Muscular myostatin gene expression and plasma concentrations are decreased in critically ill patients.

BACKGROUND The objective was to investigate the role of gene expression and plasma levels of the muscular protein myostatin in intensive care unit-acquired weakness (ICUAW). This was performed to evaluate a potential clinical and/or pathophysiological rationale of therapeutic myostatin inhibition. METHODS A retrospective analysis from pooled data of two prospective studies to assess the dynamics of myostatin plasma concentrations (day 4, 8 and 14) and myostatin gene (MSTN) expression levels in skeletal muscle (day 15) was performed. Associations of myostatin to clinical and electrophysiological outcomes, muscular metabolism and muscular atrophy pathways were investigated. RESULTS MSTN gene expression (median [IQR] fold change: 1.00 [0.68-1.54] vs. 0.26 [0.11-0.80]; p = 0.004) and myostatin plasma concentrations were significantly reduced in all critically ill patients when compared to healthy controls. In critically ill patients, myostatin plasma concentrations increased over time (median [IQR] fold change: day 4: 0.13 [0.08/0.21] vs. day 8: 0.23 [0.10/0.43] vs. day 14: 0.40 [0.26/0.61]; p < 0.001). Patients with ICUAW versus without ICUAW showed significantly lower MSTN gene expression levels (median [IQR] fold change: 0.17 [0.10/0.33] and 0.51 [0.20/0.86]; p = 0.047). Myostatin levels were directly correlated with muscle strength (correlation coefficient 0.339; p = 0.020) and insulin sensitivity index (correlation coefficient 0.357; p = 0.015). No association was observed between myostatin plasma concentrations as well as MSTN expression levels and levels of mobilization, electrophysiological variables, or markers of atrophy pathways. CONCLUSION Muscular gene expression and systemic protein levels of myostatin are downregulated during critical illness. The previously proposed therapeutic inhibition of myostatin does therefore not seem to have a pathophysiological rationale to improve muscle quality in critically ill patients. TRIAL REGISTRATION ISRCTN77569430 -13th of February 2008 and ISRCTN19392591 17th of February 2011

The role of PKD1 in aging-related neurodegeneration: Structural and functional imaging studies

Trabajo presentado en el Second Spanish Molecular Imaging Network (SMIN) Meeting, celebrado en Madrid (España) el 26 de febrero de 2018

Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D

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Neuroprotective role of PKD1 against ischemic and kainic acid-induced brain injury

Trabajo presentado en el Second Spanish Molecular Imaging Network (SMIN) Meeting, celebrado en Madrid (España) el 26 de febrero de 2018

Energy Metabolites as Biomarkers in Ischemic and Dilated Cardiomyopathy

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10−6 ). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-kappa B/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-kappa B/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders

Molecular mechanisms of myocardial remodeling

Herzinsuffizienz ist eine der Haupttodesursachen in den entwickelten Industrienationen und ihre Inzidenz und Prävalenz nimmt stetig zu. Hauptrisikofaktoren für die Entstehung einer Herzinsuffizienz sind myokardiale Umbauprozesse, die durch arterielle Hypertonie, Koronare Herzerkrankung und Klappenfunktionsstörungen hervorgerufen werden sowie durch myozytäre Hypertrophie und Fibrose gekennzeichnet sind. Die molekularen Mechanismen, welche myokardiales Remodeling verursachen und die Progression in die Herzinsuffizienz fördern, sind bislang nur unzureichend bekannt. Klinische Studien zeigten, dass die pharmakologische Hemmung des Renin-Angiotensin- Aldosteron-Systems (RAAS) und des sympathischen Nervensystems (SNS) das Überleben herzinsuffizienter Patienten verlängert. Dennoch ist die Morbidität und Mortalität von Patienten mit Herzinsuffizienz sehr hoch, sodass neue Therapieansätze dringend benötigt werden. Untersuchungen der molekularen Mechanismen des kardialen Remodelings und seiner Progression in die Herzinsuffizienz könnten dazu beitragen. Die Mechanismen myokardialer Umbauprozesse werden im Tier- und Zellkulturmodel intensiv untersucht. Es ist jedoch unklar, ob diese Befunde direkt auf Patienten übertragen werden können. Wir haben deshalb die molekularen Mechanismen von myokardialem Remodeling und Herzinsuffizienz im menschlichen Herzen untersucht. Wir fanden das kardiale RAAS und myokardiale Wachstumsfaktoren bei Patienten mit Aortenklappenstenose, Aortenklappeninsuffizienz und Dilatativer Kardiomyopathie aktiviert, wodurch interstitielle myokardiale Fibrose hervorgerufen wird. Diese molekularen und morphologischen Veränderungen traten schon sehr frühzeitig im Krankheitsverlauf auf, nahmen mit Krankheitsprogression zu und waren eng mit kardialer Funktionseinschränkung assoziiert, was zumindest teilweise die molekulare Basis der kardioprotektiven Effekte von RAAS-Inhibitoren sein könnte. Darüber hinaus wiesen wir eine Fehlregulation matrixumbauender Enzyme im hypertrophierenden und insuffizienten menschlichen Herzen nach. Die Beziehung zwischen myokardialer Fibrose und kardialer Funktionseinschränkung führte zur Annahme, dass eine Fibrosehemmung die kardiale Funktion verbessert. Wir testeten und bestätigten diese Hypothese in einem Tiermodell der kardialen Hypertrophie. Eine Hemmung von myokardialer Fibrose in der Therapie myokardialer Umbauprozesse könnte dementsprechend sinnvoll sein. Die molekularen Mechanismen, wie RAAS und sein Effektorpeptid Angiotensin-II (AngII) zu kardialen Umbauprozessen führen, sind nicht bekannt. Es war daher das Ziel, die Signaltransduktionskaskade, welche AngII bei myokardialen Umbauprozessen aktiviert, näher zu untersuchen. Von besonderem Interesse hierbei waren die Ergebnisse anderer Arbeitsgruppen, die zeigten, dass AngII die Serin/Threonin Kinase Protein- Kinase D1 (PKD1) in glatten Gefäβmuskelzellen zeit- und dosisabhängig aktiviert1, da PKD1 die transkriptionellen Repressoren Klasse-II-Histondeazetylasen (HDAC) phosphoryliert und inaktiviert. PKD1 hemmt so die Repression des prohypertrophen Transkriptionsfaktors MEF2 durch Klasse-II-HDAC. Eine zentrale Rolle dieser MEF2/Klasse-II-HDAC Regulationsachse in der kardialen Hypertrophie und der Herzinsuffizienz wurde bereits nachgewiesen2,3. Allerdings war die Bedeutung des übergeordneten Regulators PKD1 in der Kontrolle der MEF2/Klasse-II-HDAC Achse bei kardialem Remodeling in vivo unklar. Zur Untersuchung der Rolle von PKD1 in AngII-vermittelten myokardialen Umbauprozessen generierten wir eine kardiomyozytenspezifische PKD1-Keimbahndeletion und wiesen damit eine Reduktion des myokardialen Remodeling nach. Während unserer Untersuchungen zeigte sich, dass der posttranslationellen Modifikation von Proteinen und der Steuerung des Gleichgewichtes von Proteinsynthese und -abbau im myokardialen Remodeling eine zentrale Bedeutung zukommt. Diese Prozesse werden unter anderem durch die E3-Ubiquitinligasen Muscle-RING-Finger 1 und 3 (MuRF1 und 3), die spezifisch im Herzen und Skelettmuskel exprimiert werden, reguliert. Wir postulierten, dass der durch MuRF1 und 3 vermittelte Abbau von strukturellen Proteinen für das myokardiale Remodeling bedeutsam ist und bestätigten diese Hypothese durch die Verwendung von Mäusen mit einer Keimbahndeletion für MuRF1 und 3, die verschiedenen kardialen Stresssituationen ausgesetzt wurden. Die spezifischen Funktionen von MuRF1 und 3 bei myokardialem Remodeling sind Gegenstand weiterer Untersuchungen. Myokardialer Umbau stellt einen multifaktoriellen Prozess dar, welcher durch neuroendokrine Aktivierung, transkriptionelle und posttranslationelle Regulation sowie Proteinabbau reguliert wird. Die Therapie von myokardialem Remodeling und dessen Übergang in die Herzinsuffizienz erfordert daher einen multimodalen Ansatz.The prevalence of heart failure is increasing worldwide, and most people with heart failure will die or be disabled as a consequence of cardiac remodeling, such as hospitalization and sudden cardiac death due to arrhythmias. Cardiac remodeling, the leading cause of heart failure, is characterized by myocyte hypertrophy and myocardial fibrosis and caused by arterial hypertension, coronary artery disease and valvular malfunction. However, the molecular mechanisms causing myocardial remodeling and its progression towards heart failure are only beginning to be understood. Clinical studies showed an improved survival of patients with left ventricular dysfunction and heart failure receiving inhibitors of the renin-angiotensin-aldosterone-system (RAAS) and sympathetic nervous system. However, despite advances in heart failure therapy morbidity and mortality remains high. Uncovering molecular mechanisms of cardiac remodeling and its progression towards heart failure will provide novel therapeutic targets to treat this life threatening disease. Although molecular mechanisms of heart failure are intensively studied in animal models and cell culture only few human data are available. Therefore, we first focused on the molecular mechanisms of cardiac remodeling and heart failure in the human heart. Importantly, using myocardial tissue of patients with aortic stenosis, aortic regurgitation and dilative cardiomyopathy we found an activation of the cardiac RAAS, upregulation of myocardial growth factors and occurrence of myocardial fibrosis. These molecular and morphological changes were dependent on the disease stage and had impact on cardiac function underlining the clinical evidence about cardioprotective effects of RAAS inhibition. Most importantly, these molecular changes were already observed when cardiac function was still normal indicating that treatment of early stages of cardiac remodeling in the absence of cardiac dysfunction could add therapeutic benefit for affected patients. A dysregulation of matrix remodeling enzymes mainly leading to inhibition of matrixmetalloproteinases was responsible, at least partially, for myocardial fibrosis in human hearts following chronic pressure overload due to aortic stenosis. Having elucidated that myocardial remodeling was associated with decreased systolic and diastolic cardiac function we hypothesized that inhibition of cardiac fibrosis or its main component cross-linking of collagenfibrills could lead to an improved cardiac function. This hypothesis was investigated and confirmed in an animal model of cardiac hypertrophy further supporting the clinical evidence that reverse remodeling could have beneficial effects on the progression of heart failure. Although, RAAS activation has been linked to cardiac remodeling only few data linking its effector peptide angiotensin II (AngII) and transcriptional regulation are available. In this regard, AngII has been shown to mediate its effects through protein kinase D1 (PKD1) which in turn phosphorylates the transcriptional repressors class II histone deacetylases (HDAC) releasing repression form prohypertrophic / remodeling transcription factor MEF2. To investigate the role of PKD1 for AngII mediated cardiac remodeling we generated cardiomyocyte specific knockout mice and showed that deletion of PKD1 in cardiomyocytes inhibits AngII induced myocardial remodeling. Our results showed that posttranslational modification of proteins and the balance between protein synthesis and degradation are important for myocardial remodeling. These processes are regulated by the key enzymes of the ubiquitin proteasome pathway Muscle RING-finger 1 and 3 (MuRF1 and 3) which are specifically expressed within the heart and skeletal muscle. We hypothesized that MuRF1 and 3 mediated degradation of structural proteins is important for myocardial remodeling and confirmed our assumption by applying different stress stimuli to MuRF1 and 3 knockout mice. In summary, cardiac remodeling is mediated through a variety of mechanisms involving neuroendocrine activation, transcription, posttranslational regulation and protein turnover. In order to treat cardiac remodeling and its transition towards heart failure comprehensively novel therapeutic approaches need to target these different pathways