Baseline MRI Examination in the NAKO Health Study­Findings on Feasibility, Participation and Dropout Rates, Comfort, and Image Quality.

BACKGROUND: Magnetic resonance imaging (MRI) yields important information on the development and current status of many different diseases. Whole-body MRI was accordingly made a part of the multicenter, population-based NAKO Health Study. The present analysis concerns the feasibility of the baseline MRI examination and various aspects of quality assurance over the period 2014-2019. METHODS: 32 252 participants in the NAKO Health Study, aged 20 to 74, who had no contraindication to MRI were invited to undergo scanning in one of five MRI study centers across Germany. The whole-body MRI scan took about one hour and consisted of sequences for the visualization of structural and functional features of the brain, musculoskeletal system, cardiovascular system, and thoracoabdominal system. A comprehensive quality-assurance assessment was carried out, with evaluation of adverse events, the completeness of the MRI protocols, the participants' subjective perceptions, and image quality. RESULTS: 31 578 participants (97.9%) were successfully included in the MRI study. They reported a high level of comfort and suffered no severe adverse events; mild adverse events occurred in only four participants. Depending on the imaging sequence, the image quality was rated as excellent in 80.2% to 96.8% of cases. Quality assessment with respect to structural features of the brain revealed high consistency across study centers, as well as with regard to age-and sex-based differences in brain volume (men, 1203.81 ± 102.06 cm³; women, 1068.10 ± 86.69 cm³). CONCLUSION: Whole-body MRI was successfully implemented in the NAKO baseline examination and was associated with high patient comfort and very good image quality. The imaging biomarkers of the brain confirmed previously observed differences based on age and sex, underscoring the feasibility of data pooling.

Metabolic remodeling in cardiac hypertrophy and heart failure with reduced ejection fraction occurs independent of transcription factor EB in mice.

Background: A metabolic shift from fatty acid (FAO) to glucose oxidation (GO) occurs during cardiac hypertrophy (LVH) and heart failure with reduced ejection fraction (HFrEF), which is mediated by PGC-1α and PPARα. While the transcription factor EB (TFEB) regulates the expression of both PPARGC1A/PGC-1α and PPARA/PPARα, its contribution to metabolic remodeling is uncertain. Methods: Luciferase assays were performed to verify that TFEB regulates PPARGC1A expression. Cardiomyocyte-specific Tfeb knockout (cKO) and wildtype (WT) male mice were subjected to 27G transverse aortic constriction or sham surgery for 21 and 56 days, respectively, to induce LVH and HFrEF. Echocardiographic, morphological, and histological analyses were performed. Changes in markers of cardiac stress and remodeling, metabolic shift and oxidative phosphorylation were investigated by Western blot analyses, mass spectrometry, qRT-PCR, and citrate synthase and complex II activity measurements. Results: Luciferase assays revealed that TFEB increases PPARGC1A/PGC-1α expression, which was inhibited by class IIa histone deacetylases and derepressed by protein kinase D. At baseline, cKO mice exhibited a reduced cardiac function, elevated stress markers and a decrease in FAO and GO gene expression compared to WT mice. LVH resulted in increased cardiac remodeling and a decreased expression of FAO and GO genes, but a comparable decline in cardiac function in cKO compared to WT mice. In HFrEF, cKO mice showed an improved cardiac function, lower heart weights, smaller myocytes and a reduction in cardiac remodeling compared to WT mice. Proteomic analysis revealed a comparable decrease in FAO- and increase in GO-related proteins in both genotypes. A significant reduction in mitochondrial quality control genes and a decreased citrate synthase and complex II activities was observed in hearts of WT but not cKO HFrEF mice. Conclusions: TFEB affects the baseline expression of metabolic and mitochondrial quality control genes in the heart, but has only minor effects on the metabolic shift in LVH and HFrEF in mice. Deletion of TFEB plays a protective role in HFrEF but does not affect the course of LVH. Further studies are needed to elucidate if TFEB affects the metabolic flux in stressed cardiomyocytes.

Stimulation of soluble guanylyl cyclase (sGC) by riociguat attenuates heart failure and pathological cardiac remodelling.

BACKGROUND AND PURPOSE: Heart failure is associated with an impaired NO-soluble guanylyl cyclase (sGC)-cGMP pathway and its augmentation is thought to be beneficial for its therapy. We hypothesized that stimulation of sGC by the sGC stimulator riociguat prevents pathological cardiac remodelling and heart failure in response to chronic pressure overload. EXPERIMENTAL APPROACH: Transverse aortic constriction or sham surgery was performed in C57BL/6N mice. After 3 weeks of transverse aortic constriction when heart failure was established, animals receive either riociguat or its vehicle for 5 additional weeks. Cardiac function was evaluated weekly by echocardiography. Eight weeks after surgery, histological analyses were performed to evaluate remodelling and the transcriptome of the left ventricles (LVs) was analysed by RNA sequencing. Cell culture experiments were used for mechanistically studies. KEY RESULTS: Transverse aortic constriction resulted in a continuous decrease of LV ejection fraction and an increase in LV mass until week 3. Five weeks of riociguat treatment resulted in an improved LV ejection fraction and a decrease in the ratio of left ventricular mass to total body weight (LVM/BW), myocardial fibrosis and myocyte cross-sectional area. RNA sequencing revealed that riociguat reduced the expression of myocardial stress and remodelling genes (e.g. Nppa, Nppb, Myh7 and collagen) and attenuated the activation of biological pathways associated with cardiac hypertrophy and heart failure. Riociguat reversed pathological stress response in cultivated myocytes and fibroblasts. CONCLUSION AND IMPLICATIONS: Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Riociguat attenuates the changes in left ventricular proteome and microRNA profile after experimental aortic stenosis in mice.

BACKGROUND AND PURPOSE: Development and progression of heart failure involve endothelial and myocardial dysfunction as well as a dysregulation of the NO-sGC-cGMP signalling pathway. Recently, we reported that the sGC stimulator riociguat has beneficial effects on cardiac remodelling and progression of heart failure in response to chronic pressure overload. Here, we examined if these beneficial effects of riociguat were also reflected in alterations of the myocardial proteome and microRNA profiles. EXPERIMENTAL APPROACH: Male C57BL/6N mice underwent transverse aortic constriction (TAC) and sham-operated mice served as controls. TAC and sham animals were randomised and treated with either riociguat or vehicle for 5 weeks, starting 3 weeks after surgery, when cardiac hypertrophy was established. Afterwards, we performed mass spectrometric proteome analyses and microRNA sequencing of proteins and RNAs, respectively, isolated from left ventricles (LVs). KEY RESULTS: TAC-induced changes of the LV proteome were significantly reduced by treatment with riociguat. Bioinformatics analyses revealed that riociguat improved TAC-induced cardiovascular disease-related pathways, metabolism and energy production, for example, reversed alterations in the levels of myosin heavy chain 7, cardiac phospholamban and ankyrin repeat domain-containing protein 1. Riociguat also attenuated TAC-induced changes of microRNA levels in the LV. CONCLUSION AND IMPLICATIONS: The sGC stimulator riociguat exerted beneficial effects on cardiac structure and function during pressure overload, which was accompanied by a reversal of TAC-induced changes of the cardiac proteome and microRNA profile. Our data support the potential of riociguat as a novel therapeutic agent for heart failure.

Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes.

Protection achieved by ischemic preconditioning is dependent on A(2B) adenosine receptors (A(2B)AR) in rabbit and mouse hearts and, predictably, an A(2B)AR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A(2B)AR. The present study tested whether A(2B)AR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A(2B)AR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A(2B)AR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A(2B)AR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A(2B)AR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A(2B)AR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A(2B)AR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A(2B)AR band. Thus, the present study demonstrates that activation of A(2B)AR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A(2B)AR are expressed in individual cardiomyocytes. However, surprisingly, A(2B)AR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A(2B)AR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

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.

Dynamic adaptation of myocardial proteome during heart failure development.

Heart failure (HF) development is characterized by huge structural changes that are crucial for disease progression. Analysis of time dependent global proteomic adaptations during HF progression offers the potential to gain deeper insights in the disease development and identify new biomarker candidates. Therefore, hearts of TAC (transverse aortic constriction) and sham mice were examined by cardiac MRI on either day 4, 14, 21, 28, 42, and 56 after surgery (n = 6 per group/time point). At each time point, proteomes of the left (LV) and right ventricles (RV) of TAC and sham mice were analyzed by mass spectrometry (MS). In TAC mice, systolic LV heart function worsened from day 4 to day 14, remained on a stable level from day 14 to day 42, and showed a further pronounced decline at day 56. MS analysis identified in the LV 330 and in RV 246 proteins with altered abundance over time (TAC vs. sham, fc≥±2). Functional categorization of proteins disclosed the time-dependent alteration of different pathways. Heat shock protein beta-7 (HSPB7) displayed differences in abundance in tissue and serum at an early stage of HF. This study not only provides an overview of the time dependent molecular alterations during transition to HF, but also identified HSPB7 as a novel blood biomarker candidate for the onset of cardiac remodeling.

Proteome analysis of heart biopsies using a TRIzol-based protein extraction.

BACKGROUND: When dealing with rare samples of which only minute amounts are available, e.g. human heart tissue, simultaneous extraction of DNA, RNA, and proteins from the same sample is crucial for a comprehensive analysis on the physiological or pathological state of such precious tissue. In this study we provethe efficacy of a modified TRIzol protocol to extract proteins from samples of small size, such as endomyocardial biopsies (EMBs). METHOD: Initially, we compared TRIzol protein extraction efficacy to urea/thiourea extraction from total murine left ventricles and then small amounts of left and right murine ventricles. Finally, we applied the modified TRIzol protocol to the proteomic profiling of EMBs from human left and right ventricles. RESULTS: Analysis of the proteins extracted from mouse and human samples revealed sufficient protein amount for downstream applications. Thus, LC-tandem mass spectrometry permitted highly sensitive protein identifications and comparable protein patterns and coverage of cellular components as a standard extraction protocol. 2D gel-based analysis confirmed the high quality and reproducibility of the TRIzol derived protein extracts. CONCLUSION: Our results prove the utility of the modified TRIzol protocol for proteomics analyses involving minute amounts of precious samples.