MicroRNA-1 (miRNA-1) has been long viewed as a muscle-specific miRNA and plays a critical role in myocardium and cardiomyocytes by controlling myocyte growth and rhythm. We identified that miRNA-1 is expressed in cardiac fibroblasts, which are one of the major non-muscle cell types in myocardium and are responsible for cardiac fibrosis in pathological conditions. In this study, we aimed to investigate the effect and mechanism of action of miRNA-1 on cardiac fibroblast proliferation. Subcutaneous angiotensin II (Ang II) infusion via osmotic minipumps for 4 weeks was used to induce myocardial interstitial fibrosis in male Sprague-Dawley rats. MiRNA-1 expression was significantly down-regulated by 68% in freshly isolated ventricular fibroblasts from Ang II-infused rats than that from control rats. Similar results were obtained in adult rat ventricular fibroblasts that were stimulated in culture by Ang II or TGFβ for 48 h. Functionally, overexpression of miRNA-1 inhibited fibroblast proliferation, whereas knockdown of endogenous miRNA-1 increased fibroblast proliferation. We then identified and validated cyclin D2 and cyclin-dependent kinase 6 (CDK6) as direct targets of miRNA-1 in cardiac fibroblasts using biochemical assays. Moreover, we showed that the inhibitory effects of miRNA-1 on cardiac fibroblast proliferation can be blunted by overexpression of its target, cyclin D2. In conclusion, our findings demonstrate miRNA-1 expression and regulation in adult ventricular fibroblasts, where it acts as a novel negative regulator of adult cardiac fibroblast proliferation that is at least partially mediated by direct targeting of two cell cycle regulators. Our results expand the understanding of the regulatory roles of miRNA-1 in cardiac cells (i.e., from myocytes to a major non-muscle cells in the heart).

Contact sites between the mitochondria and endoplasmic reticulum (ER) are irregulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Mitofusin 2 (Mfn2) has been shown as one of the major components tethering these two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified to modulate canonical (i.e., mitochondrial fusion) and non-canonical functions, such as mitophagy and activation of ER stress signaling, however there is little information whether any PTMs can regulate mitochondrial and ER tethering. Basal tyrosine phosphorylation of Mfn2 was detected by mass spectroscopy, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases. Here we show that the mitochondria-localized Src family tyrosine kinases including c-Src can phosphorylate Mfn2, which decreases distance between the mitochondria and ER, and increases Ca2+ transfer from the ER to mitochondria, followed by changes in ROS generation and mitochondrial bioenergetics. Our findings suggest that tyrosine phosphorylation of Mfn2 may uniquely work to fine-tune ER-mitochondrial Ca2+ transport under physiological conditions, without activating mitophagy or ER stress signaling.

Introduction Contact sites between the endoplasmic reticulum (ER) and mitochondria (i.e., mitochondria‐associated membranes: MAMs) have important roles for the exchange of lipids, Ca2+, and reactive oxygen species (ROS), and greatly influence mitochondrial bioenergetics and cell fate. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, is critical for MAM formation by tethering two organelles together to initiate contact. Although several post‐translational modifications (PTMs) of Mfn2 have been identified, including serine/threonine phosphorylation and ubiquitination, it remains unclear whether the PTMs of Mfn2 regulate its tethering function. In addition, while basal tyrosine phosphorylation (P‐Tyr) of Mfn2 was reported from mass spectroscopy data, the signaling pathways that regulate P‐Tyr levels of Mfn2 are completely unknown. Objective To determine whether P‐Tyr of Mfn2 modulates MAM functions. Methods Biochemical (mitochondrial fractionation), cell biological (Foster resonance energy transfer [FRET] efficiency between the outer mitochondrial membrane (OMM)‐targeted cyan fluorescent protein and ER membrane‐targeted yellow fluorescent protein), and physiological (imaging of mitochondrial Ca2+ [mtCa2+], ROS, and membrane potential [Δψm] in live cells) assays were performed in HEK293T cells. Results Endogenous expression of several tyrosine kinases, including proto‐oncogene tyrosine protein kinase (Src), C‐Terminal Src Kinase (CSK), and proline‐rich tyrosine kinase 2 (Pyk2), was found in the cytosolic and mitochondrial fractions of HEK293T cells. Overexpression of these proteins increased P‐Tyr levels of Mfn2, as detected by a general P‐Tyr antibody. Next, we found that CSK knockdown by shRNA in HEK293T cells enhances the physical coupling between the OMM and ER membrane compared to control cells, as determined by biochemical and live‐cell FRET assays. We also found that CSK knockdown induces mild, but significant, Δψm depolarization and increases basal mitochondrial ROS levels, which were quantified by a Δψm‐sensitive dye TMRE, and a mitochondria‐targeted H2O2 biosensor mt‐RoGFP2‐Orp1, respectively. Lastly, we observed mtCa2+ uptake in response to ER Ca2+ release induced by Gq protein‐coupled receptor stimulation, using a mitochondria‐targeted Ca2+ biosensor mt‐RCamp1h. Importantly, CSK‐knockdown enhanced mtCa2+ uptake in cells compared to control despite the mild Δψm depolarization. Conclusion Mfn2 has potential to be phosphorylated by tyrosine kinases in situ. P‐Tyr levels of Mfn2 may modulate the physical coupling and Ca2+ transport between organelles, which promotes mtCa2+‐dependent Δψm depolarization and mitochondrial ROS generation. Support or Funding Information A part of this research was supported by American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

Remodeling is a common feature in cardiac disease, which entails fibroblast activation and often leads to fibrosis. MicroRNAs (miRs) are novel regulators in cardiac remodeling. In contrast to myocytes, very little is known about miRs in cardiac fibroblasts. In this study, we established the miR expression profile in adult rat ventricular fibroblasts and assessed their dynamic changes upon fibroblast activation, using high throughput TaqMan miR arrays and a culture model that mimics the phenotypic and functional changes of cardiac fibroblasts in the diseased heart. Among 518 miRs included in the arrays, 180 miRs were detected and 52 miRs were more than 4‐fold up‐ or down‐regulated upon fibroblast activation. Cardiomyocyte‐restricted miR‐208 was undetectable, indicating lack of myocyte contamination. MiR‐21, a well‐studied miR in cardiac fibroblasts and fibrosis development, was increased 3.1‐fold upon fibroblast activation, which is consistent with a 5‐fold up‐regulation reported in fibroblasts from failing hearts. We identified several previously unidentified miRs in cardiac fibroblasts. One of them is miR‐1, which has been extensively studied in myocytes. Using gain‐ and loss‐of function approaches and 3′UTR luciferase assays, we detected a novel role for miR‐1 as negative regulator of fibroblast proliferation via a mechanism that involves direct targeting of key cell cycle regulators (cyclin D1, cyclin D2 and CDK6). Together, our data suggest dynamic regulation of miR‐1 and other miRs in cardiac fibroblasts that regulate fibroblast activation and function. (The work is supported by an IDeA grant P20GM103652 from the NIH/NIGMS)