Choice of Cell-Delivery Route for Skeletal Myoblast Transplantation for Treating Post-Infarction Chronic Heart Failure in Rat

Intramyocardial injection of skeletal myoblasts (SMB) has been shown to be a promising strategy for treating post-infarction chronic heart failure. However, insufficient therapeutic benefit and occurrence of ventricular arrhythmias are concerns. We hypothesised that the use of a retrograde intracoronary route for SMB-delivery might favourably alter the behaviour of the grafted SMB, consequently modulating the therapeutic effects and arrhythmogenicity.Three weeks after coronary artery ligation in female wild-type rats, 5x10(6) GFP-expressing SMB or PBS only (control) were injected via either the intramyocardial or retrograde intracoronary routes. Injection of SMB via either route similarly improved cardiac performance and physical activity, associated with reduced cardiomyocyte-hypertrophy and fibrosis. Grafted SMB via either route were only present in low numbers in the myocardium, analysed by real-time PCR for the Y-chromosome specific gene, Sry. Cardiomyogenic differentiation of grafted SMB was extremely rare. Continuous ECG monitoring by telemetry revealed that only intramyocardial injection of SMB produced spontaneous ventricular tachycardia up to 14 days, associated with local myocardial heterogeneity generated by clusters of injected SMB and accumulated inflammatory cells. A small number of ventricular premature contractions with latent ventricular tachycardia were detected in the late-phase of SMB injection regardless of the injection-route.Retrograde intracoronary injection of SMB provided significant therapeutic benefits with attenuated early-phase arrhythmogenicity in treating ischaemic cardiomyopathy, indicating the promising utility of this route for SMB-delivery. Late-phase arrhythmogenicity remains a concern, regardless of the delivery route

Calcineurin splicing variant calcineurin Aβ1 improves cardiac function after myocardial infarction without inducing hypertrophy

BACKGROUND: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aβ (CnAβ) gene generates the CnAβ1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAβ1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown. METHODS AND RESULTS: We generated transgenic mice overexpressing CnAβ1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAβ1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAβ1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAβ1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation. CONCLUSIONS: Calcineurin Aβ1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAβ1 an attractive candidate for the development of future therapeutic approaches.British Heart Foundation [PG/07/020/22503]; Spanish Ministry of Science and Innovation [BFU2009-10016]; National Institutes of Health Research, Cardiovascular Biomedical Research Unit at the Royal Brompton; Hare-field NHS Foundation Trust; Imperial College; Spanish Fondo Nacional de Investigaciones Sanitarias [EIF-040545, ERG-239158, CP08/00144

Follistatin-like 3 mediates paracrine fibroblast activation by cardiomyocytes

Follistatins are extracellular inhibitors of the TGF-β family ligands including activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (FSTL3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. FSTL3 expression is elevated in patients with heart failure and is upregulated in cardiomyocytes by hypertrophic stimuli, but its role in cardiac remodelling is largely unknown. Here, we show that the production of FSTL3 by cardiomyocytes contributes to the paracrine activation of cardiac fibroblasts, inducing changes in cell adhesion, promoting proliferation and increasing collagen production. We found that FSTL3 is necessary for this response and for the induction of cardiac fibrosis. However, full activation requires additional factors, and we identify connective tissue growth factor as a FSTL3 binding partner in this process. Together, our data unveil a novel mechanism of paracrine communication between cardiomyocytes and fibroblasts that may provide potential as a therapeutic target in heart remodelling.British Heart Foundation [PG/08/084/25827]; Heart Research UK; National Institute for Health Research Cardiovascular Biomedical Research Unit at the Royal Brompton; Harefield NHS Foundation Trust; Imperial College; European Union [ERG-239158, ITN-289600]; Spanish Ministry of Science and Innovation [BFU2009-10016, CP08/00144]; Regional Government of Madrid [S2010/BMD-2321 'Fibroteam']S

The Calcineurin Variant CnAβ1 Controls Mouse Embryonic Stem Cell Differentiation by Directing mTORC2 Membrane Localization and Activation

Embryonic stem cells (ESC) have the potential to generate all the cell lineages that form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator MBNL1 promotes generation of the atypical calcineurin Aβ variant CnAβ1 in mouse ESCs (mESC). CnAβ1 has a unique C-terminal domain that drives its localization mainly to the Golgi apparatus by interacting with Cog8. CnAβ1 regulates the intracellular localization and activation of the mTORC2 complex. CnAβ1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/β-catenin signaling pathway, and defective mesoderm specification. In summary, here we unveil the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage.European Union's FP7 [CardioNext-ITN-608027, Cardio-NeT-ITN-289600]; Spanish Ministry of Science and Innovation [SAF2012-31451, CP08/00144]; Regional Government of Madrid [2010-BMD-2321]; Spanish Ministry of Economy and Competitiveness; Pro-CNIC Foundation; Severo Ochoa Center of Excellence (MINECO award) [SEV-2015-0505]S

Role and possible mechanisms of clenbuterol in enhancing reverse remodelling during mechanical unloading in murine heart failure

Aims Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a b 2 -adrenoceptor (b 2 -AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF. Methods and results Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4-6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF þ UN þ Sal or HF þ UN þ Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham þ Sal, HF þ Sal) or clenbuterol (HF þ Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Conclusion Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation-contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function

Author Correction: WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Translational control of cardiac fibrosis

Background Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. Methods Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used miRNA-and RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. To reveal post-transcriptional mechanisms in the human fibrotic heart, we then integrated our findings with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients. Results We generated nucleotide-resolution translatome data during the TGFβ1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested an extensive post-transcriptional regulatory network underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts. Conclusions We reveal widespread translational effects of TGFβ1 and define novel post-transcriptional events that control the fibroblast-to-myofibroblast transition. Regulatory networks that affect ribosome occupancy in fibroblasts are paralleled in human heart disease. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function