|Home | About | Journals | Submit | Contact Us | Français|
Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ.
The aim of this study was to develop a strategy that identified and evaluated the potential of specific miRNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo.
Using a combinatorial strategy, we identified a combination of microRNAs (miRNA) 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells, demonstrated that a single transient transfection of the microRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10 fold upon JAK inhibitor I treatment. Importantly, administration of microRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and non-cardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin.
The findings from this study provide the first proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.
Response to cardiac injury is characterized by inadequate cardiomyocyte regeneration and excessive fibrosis resulting in significant impairment of tissue structure and function. Despite major developments in cardiac regenerative medicine, repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge. Recently, landmark reports describing the reprogramming of somatic cells to induced pluripotent stem (iPS) cells have raised the possibility of using cellular reprogramming for cardiac tissue regeneration1, 2. These include directed cardiogenesis in vitro, of partially reprogrammed fibroblasts3 as well as transplantation of the ischemic heart with undifferentiated iPS cells4. However, concerns about the maturity and functional heterogeneity of stem cell-derived cardiomyocytes,5 the low survival and retention of delivered cells, as well as their potential tumorigenicity still exist6, 7. Some of these limitations could be overcome by direct reprogramming of somatic cells into cardiomyocytes obviating an intermediate pluripotent stage. Indeed, a recent report from Srivastava’s group describes the use of three cardiac transcription factors (Gata4, Mef2c and Tbx5) to reprogram mouse fibroblasts directly into cardiomyocytes8. Importantly, there is no evidence reported as yet, for direct reprogramming in vivo.
A promising alternative strategy for direct reprogramming is the use of microRNAs (miRNAs)9. The repressive actions of miRNAs on gene expression can be powerful as a single miRNA may target multiple pathways simultaneously. Chemically synthesized miRNA mimics are easily administered to cells via lipid-based transfection and exhibit low toxicity in animal models10. Moreover, the small size of a single miRNA allows the packing of multiple transcripts in the same delivery vector to increase both reprogramming efficiency and functional homogeneity of reprogrammed cells11.
In this report, we present the first evidence of miRNA-mediated direct conversion of fibroblasts to the cardiomyocyte lineage, in vitro and in vivo. Indeed, our data show that mouse fibroblasts can be directly converted to a cardiomyocyte-like phenotype in vitro by a single transient transfection using a distinct combination of miRNAs (miRNAs 1, 133, 208, and 499), enhanced further by JAK inhibitor I treatment. Reprogrammed cells express cardiomyocyte-specific genes and proteins and additionally, exhibit sarcomeric organization, spontaneous calcium oscillations and mechanical contractions characteristic of a cardiomyocyte-like phenotype. Importantly, studies using genetically-traced fibroblasts also suggest that such conversion can be achieved directly in the injured myocardium following miRNA delivery. Our work provides the first insight into the role miRNAs may play in cardiac reprogramming biology as well as a novel and potentially more efficient method of achieving cardiomyocyte regeneration in situ.
Synthetic mimics of mature microRNAs (Ambion/Applied Biosystems) were introduced into cells via transient transfection with Dharmafect1 (ThermoScientific) using standard conditions and at a final concentration of 50nM. As a negative control, we used a random sequence non-targeting mimic (negmiR) miRNA (Ambion/Applied Biosystems). Total RNA was isolated using RNeasy-96 (Qiagen) and PicoPure RNA isolation kits (Applied Biosystems). JAK inhibitor I (EMD Biosciences) treatment (final concentration 1nM) was initiated 2 days following transfection and continued daily for 5–8 days. Quantitative expression of each gene was assessed using Taqman Gene Expression Assays on the StepOnePlus Real-Time PCR System (Applied Biosystems).
Cells were fixed in 4% paraformaldehyde and stained using the following primary antibodies: α-actinin (Sigma), myosin heavy chain (Developmental Studies Hybridoma Bank, & Abcam), cardiac troponin I and T (Abcam), CD45 (BD Biosciences), Ckit (R&D), Smooth muscle actin (Sigma), Vimentin (Abcam), Ddr2 (R&D), RFP (Rockland Immunochemicals). Confocal images were captured using an LSM 510 Meta DuoScan microscope (Zeiss) and processed using LSM 5 software, version 4.2. Images of CFP expression were captured using the AxioObserver widefield fluorescence microscope (Zeiss). 3D images were reconstructed using Imaris software (Bitplane) of 20X confocal images using the microscope mentioned above. FACS sorting was performed using a FACStar Plus cytometer (BD Biosciences). All FACS samples were gated against an untransfected cell control 7–10 days post-transfection with miRNAs. The CFP+ cell population generated by FACS was first tested for the presence of Myh6 (since CFP is driven by the αMHC promoter) using qRT-PCR before evaluating for the expression of other genes.
Adult, male Fsp1Cre-tdTOMATO transgenic mice were subjected to LAD using previously published procedures 14. Lentiviral microRNAs (2×106 pfu in 40μL) were injected once at the time of injury, at two sites 2mm below site of ligation. Animals were harvested after 4 weeks, fixed, paraffin-embedded, and stained with anti-RFP (Rockland Immunochemicals) and anti-cardiac troponin T (Abcam).
Based on the potential miRNAs hold for regulating cellular and developmental processes15–18, we hypothesized that they may be capable of redirecting cardiac cell fate. In this study, we developed a strategy to identify specific miRNAs capable of reprogramming cardiac fibroblasts directly to the cardiomyocyte lineage. We selected six candidate miRNAs based on their roles in cardiac muscle development and differentiation19–24. Synthetic, chemically-modified mimics of miRNAs 1; 126-3p (126); 133a (133); 138, 206; and 208a(208) were introduced into mouse cardiac fibroblasts via transient transfection, alone and in all possible double and triple combinations with each other, resulting in a total of 41 different compositions (Figure 1a). These were initially evaluated for their reprogramming potential by screening for induction of markers of cardiac differentiation in MEFs, neonatal and adult cardiac fibroblasts at 3 and 6 days post-transfection using quantitative real-time PCR (qRT-PCR) (Figure 1a-c and Online Figure II). Markers included previously characterized cardiac reprogramming markers8 Gata4, Mef2c, Tbx5, as well as later stage mature cardiomyocytes markers such as Tnni3 (Cardiac troponin I). To ensure that our starting population of cardiac fibroblasts was not contaminated with cardiomyocytes, we subjected our preps to Percoll gradient centrifugation to remove the cardiomyocyte fraction and further, confirmed by FACS analysis, that contamination by cardiomyocytes or c-Kit+/sca-1+ progenitor cells is insignificant (0.01–0.04%, data not shown).
Our results showed that as early as 3 days post-transfection, miRNA-transfected cells exhibited a major shift in expression from fibroblastic genes such as Vimentin (Vim) and Ddr2 to cardiomyocyte-specific genes (Figure 1a). Additionally, controls (non-targeting miRNA (negmiR), mock and untransfected cells) including the transfection of fibroblast-enriched miR-21 did not activate cardiomyocyte markers. Interestingly, gene expression data were clustered into defined groups where distinct miRNA compositions appeared to regulate the expression of stage-specific markers of cardiac differentiation. This approach identified miRNA combinations that consistently induced a fibroblastic-myocyte phenotypic shift in both neonatal and adult cardiac fibroblasts (Figure 1a-c). Top candidates identified included miR-1 alone; miRs-1, 133, 206; miRs-1, 133, 208; miRs-133, 206, 208; miR-1, 138, and miRs-1, 138, 208.
To further validate these findings, we performed immunostaining on miRNA-transfected neonatal and adult cardiac fibroblasts for induced cardiomyocyte-specific markers including, myosin heavy chain (MHC), Cardiac troponin I (TNNI3), and α –ACTININ. As shown in Figure 1d & e and Online Figures III & IV, immunostaining on adult and neonatal cardiac fibroblasts revealed that top miRNA candidates induced protein expression of MHC, TNNI3 and/or α-ACTININ as early as 6 days after transfection. Complementary experiments conducted using fibroblasts isolated from transgenic mice expressing α-myosin heavy chain-driven cyan fluorescent protein (αMHC-CFP) revealed that αMHC was activated as early as 4 days post-transfection (Online Figure V). Moreover, transient transfection of neonatal cardiac fibroblasts isolated from double transgenic mice carrying both the Fibroblast-specific promoter 1 (Fsp1)-driven Cre recombinase gene25 and a floxed tdTomato reporter26(Fsp1-Cre/tdTomato) with miRNAs resulted in co-localization of the tdTomato marker with cardiac markers TNNI3 or α-ACTININ (Figure 1f and Online Figure VI), providing strong evidence for the fibroblastic origin of miRNA-induced cardiomyocyte-like cells. No expression of TNNI3 or α-ACTININ was detected in the any of the control fibroblasts (Online Figure VII). Interestingly, as shown in Figure 1e, prominent striations were observed as early as 1 week post-transfection in TNNI3+ cells. Organization and definition of TNNI3 and α-ACTININ expression increased progressively between 2 and 4 weeks following miRNA introduction (Figure 1f and Online Figure VI). To further test the levels of cardiac markers in reprogrammed cells specifically, we sorted the CFP+ population from αMHC-CFP neonatal cardiac fibroblasts by FACS 1 week following miRNA transfection. We chose to focus on miR-1 alone and the combination miRs-1, 133, 208 due to their overall potential at cell conversion and cardiac specificity. Interestingly, inclusion of myosin-regulated miR-49927 in the miRNA combination 1, 133, 208 did not change reprogramming efficiency as measured by CFP expression (Figure 2a). However, it did seem to increase the levels of cardiomyocyte marker expression (Figure 2b). Indeed, quantitative real-time PCR analysis of the CFP+ selected cells from fibroblasts transfected with miRs-1, 133, 208, 499 revealed a significant increase in expression of the cardiac developmental marker Mef2c, and that of the late-stage marker, Tnni3. Overall the above results provide strong evidence for the capacity of miRNAs to induce expression of cardiomyocyte markers in cardiac fibroblasts.
The data thus far have demonstrated that miRNAs are capable of inducing cardiomyocyte marker expression in fibroblasts albeit at low efficiency. Since it has been reported that small molecules might enhance cell conversion/differentiation, we decided to investigate their effects on miRNA-induced cardiac reprogramming17, 28, 29. Interestingly Efe and colleagues3 subjected partially reprogrammed mouse embryonic fibroblasts with sequential addition of JAK inhibitor I and BMP4 treatment to activate the cardiac progenitor program which yielded beating cardiomyocytes within three weeks. To evaluate the potential of this approach neonatal cardiac fibroblasts from αMHC-CFP mice were treated with miRNAs and JAK inhibitor I. Cells were assayed by FACS for enhancement of a CFP+ cell population over transfection with miRNAs alone. Transfection with these miRNA compositions resulted in induction of CFP expression in approximately 1.5–7.7% of fibroblasts (Figure 2a). However, additional treatment with JAK inhibitor I dramatically shifted the profile of CFP+ cells (up to ~28% of CFP+ cells) and increasing the efficiency of reprogramming approximately 8–10 fold (Figure 2a & c). None of the other compounds tested seemed to enhance miRNA-induced reprogramming efficiency (data not shown). Remarkably, the combination of miRs-1,133, 208, 499 plus JAK inhibitor I treatment also led to a significant elevation of ion channel expression in CFP+ cells resulting in an almost 10-fold increase in expression of the L-type calcium channel Cacna1c, when compared to non-JAK inhibitor I-treated miRNA-transfected cardiac fibroblasts (Figure 2d and Online Figure VIII).
To further validate the functional consequences of the above observations, we analyzed calcium signaling, excitation-contraction (EC) coupling and spontaneous contraction. CFP+ cells were treated with miRNA combinations versus control, all in the presence of JAK inhibitor I, for up to 4 weeks post-FACS (Figure 3). Spontaneous contraction was observed as early as 10 days in culture however this was sporadic (1–2% of total cell population). Rhythmic calcium oscillations were also often observed in miRNA transfected cells by 14 days post-FACS, but only rarely observed in control cells (negmiR and untransfected fibroblasts). Both the incidence and oscillation frequency of spontaneous calcium oscillations in miRNA-transfected fibroblasts increased with time in culture (not shown), suggesting an increase in the state of maturation. The average oscillation frequencies were significantly faster in CFP+ cells derived from fibroblasts transfected with miR-1 and miRs-1, 133, 208, 499 (1.31±0.31 oscillations/min; range: 0.15–4 oscillations/min and 1.54±0.14 oscillations/min; range: 0.1–3.15 oscillations/min, respectively) than in controls (0.45 and 0.6 oscillations/min), and reached about 50% of the average frequency observed in cultured neonatal cardiomyocytes (2.93±0.13 oscillations/min; range: 1–12 oscillations/min) (Figure 3a and b). There was also a substantial increase in the number of cells exhibiting oscillations in CFP+ reprogrammed cells (Figure 3c). Moreover, both the average oscillation frequency and the number of oscillating cells detected were greater in CFP+ cells derived from miR-1, 133, 208, 499-transfected fibroblasts than those transfected with miR-1 alone (Figure 3b and c), suggesting that the combination of miRNAs was more effective than miR-1 alone. To assess calcium entry through voltage-gated calcium channels, we also measured responses to membrane depolarization (Figure 3d). Consistent with the increased expression of the L-type calcium channels in CFP+ cells, depolarization with high extracellular [K+] (60 mM) produced a calcium transient or induced the onset of oscillations in CFP+ cells (Figure 2d, ,3d3d and Online Figure VII), whereas control cells rarely exhibited a calcium response (Figure 3e). This suggests that miRNA-transfected fibroblasts develop the cellular machinery of EC coupling, a hallmark of functional cardiomyocytes. Together, these findings provide functional support for reprogramming of miRNA-transfected fibroblasts towards a cardiomyocyte phenotype, albeit less fully matured, as evidenced by the presence of spontaneous calcium oscillations and EC coupling, and further suggest that calcium entry through voltage-gated calcium channels plays a role in regulating calcium oscillations in these cells.
Additionally, to explore the route of cardiac reprogramming, we assessed for expression of the pluripotency markers Oct4 and Nanog immediately following miRNA introduction, at 24 hour intervals, till the induction of cardiomyocte marker expression in both neonatal and adult cells and did not observe any change in gene expression relative to experimental controls (data not shown). However, we did observe rapid induction of primitive cardiac mesodermal marker Mesp2 as early as 1 day post-transfection in the case of miRs-1, 133, 208, 499 and by day 5 in the case of miR-1 (Online Figure IX). These data, along with an observed reduction in fibroblastic markers Ddr2 and Vim (Online Figure X) suggest that reprogramming events taking place in the fibroblasts are the result of a direct switch in cell fate circumventing passage through a pluripotent precursor cell type.
To evaluate the potential of our approach in the adult heart, we injected lentivirus encoding miR-1 or a combination of four lentiviruses encoding the miR combo (miRs-1, 133, 208, 499). A lentivirus encoding a non-targeting miRNA (negmiR) was used as a negative control. Viruses were injected intramyocardially immediately following injury by permanent ligation of the left ascending coronary artery (2 injection sites 2 mm below ligation site). The recipient double transgenic Fsp1-Cre/tdTomato mice allowed us to genetically-trace the fate of endogenous cardiac fibroblasts in vivo and investigate whether they were reprogrammed to a cardiac phenotype. Four weeks after infarction and virus injection, hearts were harvested, fixed and stained for cardiac troponin T (TNNT2) and tdTomato expression (Figure 4a and b and Online Figure XI). Analysis of the infarct areas in the left ventricular myocardium of mice injected with either miR-1 or the miR combo revealed the presence of large, striated, double-positive cells (tdTomato+/TNNT2+) well-incorporated in the surrounding cardiac syncytium. These were not observed in negmiR-injected hearts (Online Figure XIV). Co-expression of tdTomato and TNNT2 was further substantiated following 3D re-construction of confocal images (Figure 4b). Moreover, cardiomyocytes were isolated 6 weeks following permanent ligation of αMHC-CFP/Fsp1-Cre/tdTomato mice that had been injected with either the miR combo or negmiR (at the time of injury). The number of CFP+/tdTomato+ cardiomyocytes was then quantified; approximately 1% of cardiomyocytes from an infarcted heart injected with the miR combo were double positive for CFP and tdTomato whereas only one double-positive cardiomyocyte was found from a negmiR-injected heart (Figure 4c). The background fluorescence of tdTOMATO in the CFP channel and vice-versa was also examined and is provided in Online Figures XII and XIII). As shown, no background signal or overlap was detected in either the dtTomato or the CFP channels.
Although Fsp1-Cre is predominantly expressed in fibroblasts, activation in other cell types is a possibility. To address this we did extensive studies using both immunocytochemistry and FACS analysis to determine co-expression of the tdTomato marker with markers for cardiomyocytes, cardiac stem cells, inflammatory and vascular cells in the injured heart but we did not observe co-expression with the above-mentioned cell types (Online Figures IV-VI). To further provide evidence and exclude the possibility of our observations being the result of cardiac differentiation from progenitor cells rather than the reprogramming of fibroblasts, we tested for the capacity of miRs-1, 133, 208, 499 to induce cardiac reprogramming in fibroblasts of extra-cardiac origin. As shown in Figure 4d and Online Figures XVII-XIX, the combination of miRs-1, 133, 208, 499 was capable of mediating cardiomyocyte induction in adult tail tip fibroblasts. As with neonatal cardiac fibroblasts, we also observed a significant elevation of %CFP induction with JAK inhibitor I treatment in reprogrammed adult tail tip fibroblasts (Online Figures XVIII and XIX). We did find, however, that cells reprogrammed from adult tail tip fibroblasts were at a less mature stage in their cardiac phenotype when compared with reprogrammed counterparts from neonatal cardiac fibroblasts (no spontaneous contraction was observed). Significantly, the use of adult fibroblasts isolated from the tails of αMHC-CFP/Fsp1Cre/tdTOMATO mice (Figure 4d) further strengthens our observation that induced cells do not originate from contaminating cardiac progenitors. Additionally, the induction of cardiomyocyte-like cells in adult tail tip fibroblasts strongly implies that reprogramming is not due to cell fusion between fibroblasts and cells of a cardiac lineage but is in fact consequential to cellular reprogramming.
In summary, we provide the first evidence that distinct miRNAs are capable of inducing conversion of fibroblasts to cardiomyocyte-like cells. Moreover, this is the first report of cardiac direct reprogramming (in this case using miRNA) in vivo, and serves as proof of concept that this distinctive, innovative approach can be used to switch cell fate and induce regeneration in the heart (obviating the use of cell transplantation methods used heretofore). Remarkably, our studies suggest that miR-1 alone is sufficient to induce cardiac reprogramming but its effects are dramatically enhanced in combination with miRNAs 133, 208, and 499 and even further with JAK inhibitor I treatment. Indeed, our analysis revealed a specific set of miRNAs (miR-1, mir 133, mir 208 and mir 499) that was efficient not only at inducing expression of cardiac markers in fibroblasts but also efficient at converting the fibroblasts into cells with functional properties characteristic of cardiomyocytes such as L type channel expression, spontaneous calcium oscillations and contractility. Interestingly, the full combination of miRNAs did not change the total number of reprogrammed cells (as measured by % CFP+ cells) but rather enhanced the maturation of converted cells. Moreover, the effectiveness of partnering miRNAs with JAK inhibitor I treatment led to a significant increase in efficiency via enhancement of both αMHC induction and expression of cardiac ion channels. While the exact molecular pathways involved in these processes remain unresolved for now, our initial studies suggest that the miRNAs directly convert fibroblasts to cells of the cardiomyocyte lineage without intermediate conversion to a pluripotent state. In addition, the exact mechanism of JAK inhibitor I remains unclear. Recently, JAK inhibitor I has been reported to promote cardiomyocyte transdifferentiation of partially reprogrammed embryonic fibroblasts via suppression of pluripotency-promoting pathways3. Whether its effects are similar in postnatal cardiac fibroblasts remains to be investigated. Indeed, this is the first report describing a significant increase in efficiency of miRNA-induced direct cardiac reprogramming using this particular small molecule and may represent a major advancement in this arena.
Importantly we provide the first evidence of a non-viral method at accomplishing efficient miRNA-mediated direct cellular reprogramming of one somatic cell to another in vitro. This alone suggests that continual upregulation of miRNAs is not necessary for reprogramming to occur and that a single transient transfection of miRNAs is sufficient for cell fate switching suggesting further that the latter may actually take place within the first days of miRNA introduction. Remarkably, we provide the first evidence that distinct miRNAs are capable of inducing direct conversion of nonmyocytes to cardiomyocyte like cells in vivo. Moreover, genetic tracing methods suggest that induced cardiomyocyte-like cells are most likely of fibroblastic origin. We acknowledge that the use of Fsp1Cre might not serve as the ideal model for genetic tracing of cardiac fibroblasts. However, our preliminary data suggesting that Fsp1 is not expressed in non-fibroblasts (i.e. stem cells, smooth muscle cells, inflammatory cells) in the injured myocardium as well as evidence of reprogramming in adult tail tip fibroblasts provides multiple lines of support for the occurrence of fibroblast reprogramming in the injured myocardium. Interestingly, dermal fibroblasts transfected with miRNAs, despite the presence of clear upregulation of cardiomyocyte markers, rarely exhibited induced calcium transients indicating that the reprogrammed phenotype is at a less mature stage when compared with cardiac fibroblasts as the originating cell. This is in line with the experience of others in the reprogramming field who have also acknowledged challenges with reprogramming/driving tail tip fibroblasts to a functionally responsive cardiomyocyte-like phenotype8. Whether the propensity of those cells to become cardiomyocytes is correlated with their developmental origins remains to be determined. Importantly, our work also serves as proof-of-concept that it is possible to inject miRNAs directly in vivo, and achieve fibroblastic conversion to cardiomyocytes in the scar and peri-infarct areas of the heart. Significantly, these events were a result of the actions of miRNAs alone suggesting that the efficiency of reprogramming may be elevated in vivo by the dynamic cross-talk present within the microenvironment of a complex tissue versus the two-dimensional cell culture dish. Further optimization of miRNA delivery methods combined with JAK inhibitor I treatment in vivo may amplify the therapeutic implications of our work. Collectively, this study presents the first evidence of miRNA-induced direct conversion to cardiomyocytes in vitro and in vivo and demonstrates the powerful effect of this group of small RNAs at mediating cellular reprogramming.
Response to cardiac injury is usually characterized by inadequate regeneration and excessive fibrosis resulting in significant impairment of tissue structure and function. Although stem cell therapy holds promise, the data thus far suggest that authentic cardiac regeneration is modest at best. Recently, reports of reprogramming of somatic cells to inducible pluripotent stem cells or cells of other lineages have raised the possibility of using reprogrammed cells for cell therapy.. A method to directly convert the injured areas to functional tissue in vivo would be the ideal. Here, using a model of cardiac injury, we provide the first evidence that fibroblasts can be directly reprogrammed to cardiomyocytes in the heart. Our studies show that a) miRNAs are capable of converting fibroblasts to cardiomyocyte-like cells in vitro. b) miRNAs can direct in vivo conversion of fibroblasts to cardiomyocytes in the adult mouse myocardium and c) a combination of miRNAs with JAK inhibitor I treatment, significantly increases the efficiency of fibroblast reprogramming towards the cardiac fate. A therapeutic approach administering miRNAs would represent a novel and potentially simpler strategy to stimulate cardiac regeneration. This approach may have broad implications for therapeutic tissue regeneration in general.
We are very grateful to Harold Moses of Vanderbilt University, TN, for sharing his Fsp1-Cre transgenic mouse model. We are also appreciative of the input provided by all members of our lab, especially Hui Mu, Jeffrey Schmeckpeper, and Jose Gomez for technical input and helpful discussion. We also want to acknowledge James Pearson and Nicholas Barrows of the Duke RNAi Screening Facility and Mike Cook of the Duke Flow Cytometry Core Facility for their technical assistance.
SOURCES OF FUNDING
Research conducted in these studies was supported by NHLBI grants RO1 HL35610, HL81744, HL72010, and HL73219 (to V.J.D.); the Edna and Fred L. Mandel Jr Foundation (to V.J.D. and M.M.); and the Foundation Leducq (to V.J.D.). M.M. is also supported by an American Heart Association National Scientist Development Award (10SDG4280011). P.R. is supported in part by NIH grant RO1-HL093470. K.P. was supported by NIH grant HL49277.