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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.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).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.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.

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.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.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.Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression.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.

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.

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.

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.