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Fibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p

Background Given known inefficiencies in reprogramming of fibroblasts into mature induced cardiomyocytes ( iCM s), we sought to identify small molecules that would overcome these barriers to cardiac cell transdifferentiation. Methods and Results We screened alternative combinations of compounds known to impact cell reprogramming using morphologic and functional cell differentiation assays in vitro. After screening 6 putative reprogramming factors, we found that a combination of the histone deacetylase inhibitor sodium butyrate, the WNT inhibitor ICG ‐001, and the cardiac growth regulator retinoic acid (RA) maximally enhanced iCM generation from primary rat cardiac fibroblasts when combined with administration of the cardiodifferentiating transcription factors Gata4, Mef2C, and Tbx5 ( GMT ) compared with GMT administration alone (23±1.5% versus 3.3±0.2%; P GMT –sodium butyrate, ICG‐001, and RA treatment. Human iCM generation was likewise enhanced when administration of the human cardiac reprogramming factors GMT , Hand2, and Myocardin plus miR‐590 was combined with sodium butyrate, ICG‐001, and RA compared with GMT , Hand2, and Myocardin plus miR‐590 treatment alone (25±1.3% versus 5.7±0.4%; P iCM s also more frequently demonstrated spontaneous beating in coculture with neonatal cardiomyocytes with the addition of sodium butyrate, ICG‐001, and RA to transcription factor cocktails compared with transcription factor treatment alone. Conclusions The combined administration of histone deacetylase and WNT inhibitors with RA enhances rat and human iCM generation induced by transcription factor administration alone. These findings suggest opportunities for improved translational approaches for cardiac regeneration.

Nature Communications 8: Article number: 14338 (2017); Published: 6 February 2017; Updated: 13 June 2017 This Article contains errors in Figs 2 and 3, for which we apologize. In Fig. 2c, the four images were inadvertently duplicated from the images in Fig. 2b. In Fig. 3g, the image at the upper right corner, corresponding to the condition UT_ Cln3Δex7-8 was inadvertently duplicated from the image in the lower right corner of Fig.

Background: Sall4 is an essential component in maintaining embryonic stem cell self-renewal and pluripotency. Given its functional and protein level interaction with cardiac trans-differentiation factors Tbx5/Gata4, as well as its critical role in modulating embryonic cardiac development, we hypothesized that Sall4 may play an important role in cardiac reprogramming. Material/Method: We conducted gain- and loss- of function studies on neonatal and adult cardiac fibroblast from both mouse and rat. We also evaluated Sall4 function on infarcted rat heart. Results: Gata4/Mef2c/Tbx5 (GMT) administration markedly upregulated mRNA expression levels of stem factors including Sall4 , Bmi1 , and p63 , while inhibition of Sall4 largely enhanced GMT- mediated Cardiac troponin T ( cTNT ) induction. Conversely, overexpression of Sall4 plus Gata4/Mef2C in cardiac fibroblast for 7 days induces over 30-fold increase of Oct4 and Nanog by real-time PCR; longer culture induced rapidly-dividing iPSC-like clusters in both neonatal mouse and adult rat cardiac fibroblasts. Further tests showed that these iPSC-like cells were partially positive in alkaline phosphatases staining, capable of forming cardiosphere and differentiating into various cell types. Sall4 delivery also improved post-infarct cardiac function to a level that is comparable with Gata4 in rat. Conclusion: These results suggested that Sall4 is a promising target for cardiac reprogramming even though further characterization in vitro as well as in vivo is needed.

Background: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors Gata4, Mef2c and Tbx5 (GMT), and microRNAs can convert fibroblasts into iCMs, albeit with low efficiency in vitro . We aimed to increase the efficiency, maturation and speed of direct reprogramming using a unique combination of small molecules, in combination with previously identified reprogramming transcription factors, that we selected and combined in a cocktail specifically formulated to effect optimal impact on the aspects of reprogramming pathways thought to represent “roadblocks” or points of opportunity in these pathways. Methods: We screened a variety of such candidate compounds in primary rat/human cardiac fibroblast. Cardiac reprogramming was induced by lentivirus encoding Gata4, Mef2c and Tbx5 plus small molecules, and the presence of iCM was confirmed 10 days later by QRT-PCR, IFs and FACS analysis for the activation of endogenous cardiac troponin T (cTnT). Results: After validating and optimizing the dose and conditions for adding small molecules to the reprogramming cocktail, we treated GMT-overexpressing fibroblast with a combination of sodium butyrate (SB, 1mM; HDAC inhibitor), ICG-001(1μM; WNT inhibitor), and retinoic acid (RA, 1μM). We found that that a combination of SB, ICG-001, and RA significantly increased GMT-induced reprogramming efficiency to 22 % cTnT + iCMs from primary rat cardiac fibroblasts compared to 8% in the presence of single compounds and 3.5% without any added small molecule compound alone. QRT-PCR and IFs, analysis shows that a combination of the SB, ICG-001, and RA increased cTnT gene expression 4-6 fold when added to GMT-overexpressing rat cardiac fibroblasts. We also demonstrated that combined addition of SB, ICG-001, and RA increased cardiac fibroblast reprogramming efficiency 4 fold when added to GMTHM plus miR-590-overexpressing human cardiac fibroblasts. Conclusions: Thus, HDAC and WNT inhibitors along with RA, jointly accelerate GMT-induced cardiac reprogramming from rat/human cardiac fibroblasts and pave the way for new translational approaches for cardiac regeneration.

Background: The cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) have been used to successfully reprogram cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs). However their effects on other cell types remain largely unknown. Endothelial cells are an important population of the heart by Pinto et al. (2016). Given the more profound iCM induction effects observed in vivo, we have speculated that reprogramming of endogenous endothelial cells may also play an important role. Purpose: To investigate the cellular reprograming effects and mechanisms of GMT factors on cardiac endothelial cells and compare the outcomes with that from cardiac fibroblasts. Methods: Adult rat cardiac endothelial cells (REC) and cardiac fibroblasts (RCF) were purchased commercially. The cells were cultured in appropriate media and infected with GMT-expressing lentivirus and maintained with iCM media. Fourteen days later Immunofluorescence staining, FACS, and qRT-PCR were performed. Results: Both GMT treated RCFs and RECs showed positive staining of cardiac marker cTnT and αActinin-positive cells via immunofluorescence staining. FACS analysis showed GMT treated RECs had 16.7% of cTnT positive cells versus 3.3% in GMT treated RCF’s. qRT-PCR results demonstrated significant up-regulation of cTnT in GMT infected RECs (74.4±23.3 times, p Conclusion: This report suggests that RECs are susceptive to GMT induced cardiac reprogramming than in RCFs. Our study also indicate that EMT and MET sequential process are involved in the reprogramming of RECs toward iCM cells. RECs could be targeted towards iCM generation for a higher reprogramming rate.

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1–3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4–6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8. di Ronza et al. identify CLN8 as a cargo receptor for lysosomal enzymes required for their endoplasmic-reticulum-to-Golgi transport, linking Batten disease caused by CLN8 mutations to defects in organelle biogenesis.

Objective: Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown. Methods: In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes in vitro and accordingly ventricular function after myocardial infarction in rat, in vivo . Results: We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression in vitro , echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%]. Conclusions: Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs), in vitro and in vivo, and provide new insight into the molecular mechanism underlying cardiac regeneration.

Introduction: Adenovirus (Ad) vectors do not integrate into host genomes and thus offer clinical benefits as non-mutagenic, acute-term gene delivery vehicles. One limitation to Ad vector use is the transient duration of transgene expression, supposedly resulting from immune-mediated cytolysis of Ad-infected cells. Our recent observation of Ad-induced transdifferentiation of infected cardiac fibroblasts into induced cardiomyocytes over 28 days in vivo called this premise into question. Therefore, we hypothesized that Ad remains in the myocardium for over 28 days and, to our knowledge, are the first to suggest alternate mechanisms for this persistence. Methods and Results: Ad encoding green fluorescent protein (AdGFP) or mock was administered via intramyocardial injection into Sprague-Dawley rats, and Ad genome and GFP expression were analyzed 3 or 28 days later (n=6 per group, 3 euthanized at each time point). Polymerase chain reaction typing and amplicon sequencing confirmed the presence of Ad DNA in all 6 treated animals. Quantitative real time polymerase chain reaction assays further demonstrated more than a three-fold increase in Ad DNA levels at both days 3 and 28 compared to untreated control animals (3.41±1.40 and 3.37±1.23, respectively). Thus, at 28 days Ad DNA persisted at a high level comparable to that which was present at 3 days. Next, AdGFP expression and host immune response were characterized by fluorescence microscopy and histology. We found that GFP-expressing cells decreased five-fold by 28 days (49±9 v. 3±0 cells). In addition, hematoxylin and eosin staining showed minimal infiltrating mononuclear inflammatory cells in AdGFP and mock-treated animals at both time points. Conclusions: Ad DNA and infected host cell persistence suggests that host-mediated cytolysis may not be responsible for the downregulation of transgene expression, indicating that Ad vectors may consequently be useful for therapies like cellular reprogramming wherein host cell persistence is required.

The administration of a variety of reprogramming factor cocktails has now been shown to reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, reductions in ventricular fibrosis observed after reprogramming factor administration seem to far exceed the extent of induced cardiomyocyte-like cell generation in vivo. We investigated whether reprogramming factor administration might primarily play a role in activating antifibrotic molecular pathways.Adult rat cardiac fibroblasts were infected with lentivirus encoding the transcription factors Gata4, Mef2c, or Tbx5, all 3 vectors, or a green fluorescent protein control vector. Gene and protein expression assays were performed to identify relevant antifibrotic targets of these factors. The antifibrotic effects of these factors were then investigated in a rat coronary ligation model.Gata4, Mef2c, or Tbx5 administration to rat cardiac fibroblasts in vitro significantly downregulated expression of Snail and the profibrotic factors connective tissue growth factor, collagen1a1, and fibronectin. Of these factors, Gata4 was shown to be the one responsible for the downregulation of the profibrotic factors and Snail (mRNA expression fold change relative to green fluorescent protein for Snail, Gata4: 0.5 ± 0.3, Mef2c: 1.3 ± 1.0, Tbx5: 0.9 ± 0.5, Gata4, Mef2c, or Tbx5: 0.6 ± 0.2, P .05). Chromatin immunoprecipitation quantitative polymerase chain reaction identified Gata4 binding sites in the Snail promoter. In a rat coronary ligation model, only Gata4 administration alone improved postinfarct ventricular function and reduced the extent of postinfarct fibrosis.Gata4 administration reduces postinfarct ventricular fibrosis and improves ventricular function in a rat coronary ligation model, potentially as a result of Gata4-mediated downregulation of the profibrotic mediator Snail.

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Full list of SALL4-bound genes. (PDF 334Â kb)

Supplementary data. (PDF 317Â kb)

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte‐like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Methods and Results Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 ( GMT ); Hand2 (H), Myocardin (My), or microRNA (miR)‐590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte‐like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T ( cTnT ), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR‐590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH 6 and TNNT 2. When cocultured with murine cardiomyocytes, cTnT ‐expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR‐590 alone also downregulated fibroblast genes COL 1A1 and COL 3A1. miR‐590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR‐590 in inducing cellular reprogramming. Conclusions These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR‐590‐mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming.

Objective: Transdifferentiation of cardiac fibroblasts into cardiomyocyte-like cells (iCMs) represents a promising strategy in treating human heart disease. However, despite encouraging reprogramming data obtained from rodent models, the transgenes used in those models are not sufficient to reprogram human cells. We therefore sought to optimize a cocktail of factors which can efficiently transdifferentiate human cells. A porcine model was included to test its potential as a surrogate for human reprogramming studies. Methods: Lentivirus expressing Gata4 (G), Mef2c (M), Tbx5 (T), Hand2 (H), Myocardin (My), or two microRNAs miR-590, miR-199, were transduced into cultured porcine cardiac fibroblasts (PCFs) and human cardiac fibroblasts (HCFs) in different combinations. Two weeks after transduction, qRT-PCR, immunofluorescence (IF), and FACS assays were performed, and the efficiency of iCM production was evaluated by the expression of cardiomyocyte markers cardiac troponin T (cTnT) and α-sarcomeric actinin. Results: Combined G, M, T treatment alone was insufficient to transdifferentiate PCFs or HCFs into iCMs, despite the capability of GMT transduction to transdifferentiate up to 7% of treated rat cardiac fibroblasts, as assessed by FACS analysis for cTNT. However, the addition of H, My and miR-590 to GMT resulted in the transdifferentiation of approximately 5% of HCFs and PCFs, as measured by cTnT expression. IF analysis likewise demonstrated high expression of cTNT and α-actinin in these cells. Importantly, the transdifferentiated PCFs exhibited spontaneous contractions when co-cultured with murine cardiomyocytes. qPCR showed that administration of GMT plus either miR-590 or HMy upregulated cardiac genes MYH6 and TNNT2, and downregulated the fibroblast genes Collagen I and Collagen III. Mir-590 also directly suppressed Sp1, a fibrosis inducer and putative inhibitor of cellular reprogramming in the heart. Conclusions: Our data suggest that the porcine model can serve as an appropriate surrogate for human fibroblasts reprogramming studies. Enhanced transdifferentiation associated with miR-590-mediated repression of Sp1 suggests a novel pathway that may be targetable to enhance cardiac cellular reprogramming clinically.

Objective: The reprogramming of cardiac fibroblasts into induced cardiomyocytes (iCMs) improves ventricular function in myocardial infarction models. Only integrating chronic expression vectors have thus far been used to administer reprogramming genes, potentially limiting clinical applicability. We hypothesized that reprogramming could be achieved using non-integrating, acute expression adenoviral vectors. Methods: Adenoviral (Ad) and lentiviral vectors encoding Gata4 (G), Mef2c (M) and Tbx5 (T) were validated in vitro . Sprague Dawley rats then underwent coronary artery ligation and Ad-mediated administration of vascular endothelial growth factor to provide infarct prevascularization. Three weeks later, Ad or lentivirus encoding G, M, or T or an equivalent dose of a null vector was administered. Outcomes were analyzed by serial echocardiography, and by terminal MRI and histology. Results: Ad and lentivirus vectors provided equivalent in vitro GMT expression by Western blotting and AdGMT induced expression of the cardiomyocyte marker cTnT in approximately 7% of cardiac fibroblasts, compared to 4% of cells infected with LentiGMT. Sections of infarcted myocardium from rats that had been treated with AdGMT or LentiGMT demonstrated higher density of cells expressing the cardiomyocyte marker MHY7 compared to AdNull treated animals (p Conclusions: Adenoviral vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into iCMs and improving cardiac function in post-infarct rat hearts. The utility of short-term expression Ad vectors represents an important potential tool in inducing cardiac cellular reprogramming clinically.

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors.Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology.Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P .05).Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans.

Objective Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new heart failure therapy. We hypothesized that inactivation of p63 , a p53 gene family member, may help overcome human cell resistance to reprogramming. Methods p63 knockout ( -/- ) and knockdown murine embryonic fibroblasts (MEFs), p63 -/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2 and Myocardin (HM). Results Flow cytometry revealed that a significantly greater number of p63 -/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared to wild type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, p p63 -/- MEFs increased cTnT expression to 74% ± 3% of cells, but did not induce cTnT expression in WT MEFs. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT mRNA expression compared to untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout, and expressed sarcomeric α-actinin as well as cTnT. These p63 -/- adult cardiac fibroblasts exhibited calcium transients and electrically-stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing heart failure procedures. Conclusions Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.