regulate murine cardiomyocyte maturation through MRTF-SRF signaling

Yuxuan Guoa,1,2,3, Yangpo Caoa,1, Blake D. Jardina,1, Isha Sethia,b, Qing Maa, Behzad Moghadaszadehc, Emily C. Troianoc, Neil Mazumdara, Michael A. Trembleya, Eric M. Smalld, Guo-Cheng Yuanb, Alan H. Beggsc, and William T. Pua,e,2

aDepartment of Cardiology, Boston Children’s Hospital, Boston, MA 02115; bDepartment of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02215; cDivision of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115; dAab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and eHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138

Edited by Janet Rossant, The Gairdner Foundation, Toronto, ON, Canada, and approved November 24, 2020 (received for review May 6, 2020) The paucity of knowledge about cardiomyocyte maturation is a Mechanisms that orchestrate ultrastructural and transcrip- major bottleneck in cardiac regenerative medicine. In develop- tional changes in cardiomyocyte maturation are beginning to ment, cardiomyocyte maturation is characterized by orchestrated emerge. Serum response factor (SRF) is a factor that structural, transcriptional, and functional specializations that occur is essential for cardiomyocyte maturation (3). SRF directly acti- mainly at the perinatal stage. Sarcomeres are the key cytoskeletal vates key regulating assembly, electrophysiology, structures that regulate the ultrastructural maturation of other and mitochondrial metabolism. This transcriptional regulation organelles, but whether sarcomeres modulate the signal trans- subsequently drives the proper morphogenesis of mature ultra- duction pathways that are essential for cardiomyocyte maturation structural features of myofibrils, T-tubules, and mitochondria. remains unclear. To address this question, here we generated mice Sarcomeres perform a central role of converting transcriptional with cardiomyocyte-specific, mosaic, and hypomorphic mutations information into ultrastructural changes, partly by providing of α--2 (Actn2) to study the cell-autonomous roles of sarco- scaffolds for the biogenesis of T-tubules and mitochondria. Al- meres in postnatal cardiomyocyte maturation. Actn2 mutation though prior studies have suggested that sarcomeres also regulate resulted in defective structural maturation of transverse-tubules signal transduction and thereby transcription (4, 5), whether this Actn2 DEVELOPMENTAL BIOLOGY and mitochondria. In addition, mutation triggered transcrip- signaling role of sarcomeres contributes to cardiomyocyte matu- tional dysregulation, including abnormal expression of key sarco- ration was not previously evaluated. meric and mitochondrial genes, and profound impairment of the In noncardiomyocytes, the polymeric state of β-, a ubiq- normal progression of maturational expression. Mechanisti- uitous actin isoform, could regulate SRF transcription activity by Actn2 cally, the transcriptional changes in mutant cardiomyocytes modulating its cofactors myocardin-related strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates Significance cardiomyocyte maturation. Actn2 mutation increased the mono- meric form of cardiac α-actin, which interacted with the SRF cofac- tor MRTFA and perturbed its nuclear localization. Overexpression Mature cardiomyocytes are highly adapted to pump efficiently of a dominant-negative MRTFA mutant was sufficient to recapit- over billions of cycles. The mechanisms that regulate and co- ulate the morphological and transcriptional defects in Actn2 and ordinate the acquisition of the specialized features of mature Srf mutant cardiomyocytes. Together, these data indicate that cardiomyocytes in the postnatal period are not well under- Actn2-based sarcomere organization regulates structural and stood. This knowledge gap hinders studies of disease patho- – transcriptional maturation of cardiomyocytes through MRTF-SRF genesis and the maturation of stem cell derived cardiomyocytes signaling. for cardiac regenerative medicine. Here, we studied the roles of sarcomeres, the contractile machinery of cardiomyocytes, cardiomyocyte maturation | actinin 2 | MRTF | SRF | cardiomyopathy in regulating cardiomyocyte maturation. Using an in vivo genetic mosaic approach to circumvent animal lethality and secondary consequences of cardiac dysfunction, we analyzed ardiomyocyte maturation is essential for the generation of the contribution of sarcomeres to cardiomyocyte maturation Crobust cardiomyocytes that contract forcefully throughout in a physiological context. We identified an important role of postnatal life. The transcriptional, morphological, and functional sarcomeres in the regulation of signal transduction and gene hallmarks of mature cardiomyocytes have been well described (1, 2), transcription. but little is known about how cardiomyocytes acquire these features during development. This knowledge gap obscures the contribution Author contributions: Y.G., Y.C., and W.T.P. designed research; Y.G., Y.C., B.D.J., Q.M., of cardiomyocyte maturation to both cardiac disease pathogenesis B.M., E.C.T., and N.M. performed research; Y.G., B.M., E.C.T., M.A.T., E.M.S., and A.H.B. and the loss of regenerative capacity. It also impairs efforts to contributed new reagents/analytic tools; Y.G., Y.C., B.D.J., I.S., G.-C.Y., and W.T.P. ana- lyzed data; Y.G. and W.T.P. wrote the paper; and M.A.T. and E.M.S. provided MRTF mice. engineer mature stem cell–derived cardiac tissues for cell therapy, The authors declare no conflict of interest. in vitro disease modeling, and pharmacological testing. The ultrastructural hallmarks of cardiomyocyte maturation This article is a PNAS Direct Submission. include the expansion of sarcomeres, the specialized cytoskeletal Published under the PNAS license. structures that produce contractile force. In addition, the plasma 1Y.G., Y.C., and B.D.J. contributed equally to this work. membrane invaginates and forms transverse tubules (T-tubules), 2To whom correspondence may be addressed. Email: [email protected] or William.Pu@ which facilitate action potential propagation and excitation– cardio.chboston.org. 3Present address: School of Basic Medical Sciences, Institute of Cardiovascular Sciences, contraction coupling. Mitochondria grow massively in both number Peking University Health Science Center; Key Laboratory of Molecular Cardiovascular and size to enhance energy production capacity. The development Sciences, Ministry of Education, Beijing, 100191, China. of these intracellular structures is coupled with This article contains supporting information online at https://www.pnas.org/lookup/suppl/ changes to establish the contractile, electrophysiological, and met- doi:10.1073/pnas.2008861118/-/DCSupplemental. abolic functions of adult cardiomyocytes (1, 2). Published December 23, 2020.

PNAS 2021 Vol. 118 No. 2 e2008861118 https://doi.org/10.1073/pnas.2008861118 | 1of11 Downloaded by guest on October 4, 2021 A and B (MRTFA/B; also known as MKL1/2). The monomeric section) as well as ectopic splicing between exon 1 and exon 7, ΔE2-6 form (G-actin) of β-actin interacts with MRTFs and retains them which we denote as Actn2 (SI Appendix, Fig. S2 C and D). in the cytoplasm, inhibiting their transcriptional functions (6). This ectopic splicing preserved the open reading frame, and we The polymerization of β-actin into a filamentous state (F-actin), confirmed the expression of this , at a much lower level by contrast, decreases G-actin concentration, derepresses MRTFs, than ACTN2 in control mice, by Western blot using an anti- and activates SRF. body that recognizes the C terminus of ACTN2 (SI Appendix, In cardiomyocytes, F-actin is a major component of the sarco- Fig. S2E). Δ meric thin filaments and is built by the cardiac-specific isoform of ACTN2 E2-6 lacks amino acids 42 to 204 (SI Appendix, Fig. α-actin (ACTC1). The assembly and organization of ACTC1 S2F), which cover the majority of the actin-binding domains of Δ polymers into sarcomeres require α-actinin-2 (ACTN2), which ACTN2. AAV-mediated expression of ACTN2 E2-6-GFP did directly interacts with ACTC1 filaments and organizes their not detectably alter cardiomyocyte morphological maturation or alignment at Z-lines, which form the lateral boundaries of indi- overall heart function (SI Appendix, Fig. S2 G–J). Thus, Δ vidual sarcomeres. Although MRTFA/B are known to be essential ACTN2 E2-6 is a hypomorphic mutant that does not exert a in postnatal cardiomyocytes (7), whether ACTC1 polymerization dominant-negative impact on cardiomyocyte maturation.

regulates MRTF-SRF signaling has not been determined. F Technical challenges have complicated studies to understand Mosaic Actn2 Mutation in the Heart. Actn2 alleles were combined with Cre-inducible fluorescent protein (FP) reporter alleles the physiological functions of sarcomere components: in vitro Cas9GFP tdTomato cell culture artificially perturbs sarcomere organization in car- Rosa (13) or Rosa (14) so that FP reporters could diomyocytes, and organ-wide genetic manipulation of sarcomere be used as surrogate markers of AAV-Cre transduced car- diomyocytes. Full-dose (1.1 × 1010 viral genome per gram body genes often causes cardiac pathology or animal lethality. Con- F/F sequently, although many sarcomere components, including weight [vg/g]) AAV-Cre treatment of P1 Actn2 pups resulted ACTN2 and ACTC1, have been investigated by biochemical or in death about 1 wk postinjection (Fig. 1A). By echocardiogra- cell culture methods, the in vivo functions of these molecules in phy, high- (3.6 × 109 vg/g) and mid-dose (1.1 × 109 vg/g) AAV- development and homeostasis remain poorly explored. Cre triggered acute , characterized by To circumvent these issues, here we used adeno-associated diminished left ventricular systolic function and dilatation virus (AAV)-mediated cardiac genetic mosaic analysis (8, 9) to (Fig. 1B). Picro sirius red and wheat germ agglutinin (WGA) study sarcomere functions in mice. This approach circumvents staining revealed cardiac fibrosis in the high- and mid-dose animal lethality and the secondary effects of heart dysfunction, AAV-Cre–treated (Fig. 1C). Real-time qPCR detected enabling investigation of cell-autonomous functions of sarco- the up-regulation of cardiac stress markers Nppa and Nppb in mere genes within a physiological context. We hypothesized that these groups with cardiac dysfunction (Fig. 1D). These data ACTC1 polymerization regulates cardiomyocyte maturation confirmed that ACTN2 is required to maintain normal heart through MRTF-SRF signaling. To test this hypothesis, we im- function. paired ACTC1 polymerization by mutating ACTN2 and ob- Cardiac dysfunction triggers cascades of secondary effects that serving the effects on ACTC1 polymerization, MRTF-SRF confound the study of cardiomyocyte maturation (8, 9). To cir- cumvent this problem, we identified a low dose (3.6 × 108 vg/g) of signaling, and cardiomyocyte maturation. F/F AAV-Cre that recombined ∼11% of Actn2 cardiomyocytes Results without causing overall heart dysfunction, fibrosis, or activation of F/F FP/FP Cas9-Based Somatic Mutagenesis of Actn2 in Cardiomyocyte Maturation. cardiac stress markers (Fig. 1 A–D). We treated Actn2 ;Rosa We previously established a Cas9/AAV9-based somatic mutagene- mice with this dose of AAV-Cre at P1 and isolated car- sis (CASAAV) system (SI Appendix,Fig.S1A) to quickly determine diomyocytes at P30. Immunofluorescent staining of ACTN2 in the role of a given gene in cardiomyocyte maturation (8, 10). We these dissociated cardiomyocytes (Fig. 1E) showed a dramatic produced two AAV vectors targeting Actn2. Each AAV expresses decrease of ACTN2 signal in 80 to 85% of FP+ cardiomyocytes two independent guide RNAs (gRNAs) and Cre recombinase un- (Fig. 1F), while over 95% FP− cardiomyocytes retained strong der the control of the cardiomyocyte-specific cardiac T ACTN2 staining (Fig. 1F). Therefore, FPs reliably mark mutant (cTnT) (11, 12). These AAVs were injected into cardiomyocytes in this system. To characterize transcription in control and mutant cardiomyocytes, postnatal day one (P1) mice carrying a Cre-activated Cas9-2A-GFP F/F we purified FP+ cardiomyocytes from AAV-Cre–treated Actn2 (green fluorescent protein) allele (13) to trigger Cas9-based muta- F/+ genesis of Actn2. We verified successful depletion of ACTN2 pro- and Actn2 mice by fluorescence-activated cell sorting (FACS) tein in ∼60% of transduced (GFP+) cardiomyocytes at P30 by (SI Appendix,Fig.S3A) and performed real-time qPCR and immunostaining (SI Appendix,Ta1B-C).GFP+cardiomyocytes RNA sequencing (RNA-Seq) (SI Appendix,Fig.S3B). RNA-Seq exhibited defective T-tubule formation and drastically reduced analysis confirmed aberrant splicing between exons 1 and 7 and cell size (SI Appendix,Fig.S1D–F), indicating cardiomyocyte dramatically decreased expression of exons 2 to 6 in mutant maturation defects caused by ACTN2 depletion. These data en- cardiomyocytes (SI Appendix,Fig.S3C and D). Exon 1 to exon 5 couraged us to mechanistically analyze the link between ACTN2 junctions were also observed, which likely triggered nonsense- and cardiomyocyte maturation. mediated RNA decay and reduced expression of all other exons (SI Appendix,Fig.S3C and D). Overall reduction of Actn2 Generation of a Conditional Mutant Allele of Actn2. We generated a mRNA was validated by real-time qPCR using a probe tar- F floxed Actn2 allele (Actn2 ), in which the genomic region con- geting exons 11 to 12 (SI Appendix,Fig.S3B). Consistent with taining exons 2, 3, and 4 was flanked by LoxP sequences (SI these mRNA changes, the immunofluorescence signal of F/+ Δ Appendix, Fig. S2A). Heterozygous (Actn2 ) and homozygous ACTN2 E2-6 protein in the mutant cardiomyocytes was very F/F (Actn2 ) mice were fertile and exhibited normal heart function weak (Fig. 1E, right “High Exposure”) as compared to wild-type (SI Appendix, Fig. S2B). We expected that Cre-based recombi- ACTN2 in control cardiomyocytes. The low level of the mutant Δ nation of this allele would remove exons 2 to 4 and result in ACTN2 E2-6 protein makes a dominant-negative effect unlikely. joining exons 1 to 5, resulting in a frameshift mutation and re- Thus, defects in Actn2 mutant cells should be interpreted as the duction of Actn2 mRNA through nonsense-mediated decay. consequence of effective ACTN2 depletion rather than the loss F Treatment of Actn2 mice at P1 with AAV-Cre, an AAV vector of just the actin-binding domain. expressing Cre driven by the cardiomyocyte-selective cTnT Together, these data indicate that we have created a cardiac promoter (11), triggered these expected findings (see next genetic mosaic model of Actn2 mutation. This system provides an

2of11 | PNAS Guo et al. https://doi.org/10.1073/pnas.2008861118 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling Downloaded by guest on October 4, 2021 Echocardiogram ABAAV-Cre at P1 to Actn2F/F;RosaFP/FP 100 4 n=4 n=7 *** Dosage (vg/g) (0.07) (mm) 2 (0.65) 80 n=11 LVID;d 3.6x108 (Low) 0 4 *** 60 *** 1.1x109 (Mid) (0.75) 2 (mm) LVID;s 40 3.6x109 (High) 0 Survival (%) 40 *** 20 1.1x1010(Full) *** (0.92) FS

n=9 (%) 20 n=10 5 4 7 0 0 0 7 14 21 28 AAV-Cre: High High Mid Low Postnatal Day Actn2 : F/+ F/F Actn2: F/F 500 CDNppa AAV-Cre: High Mid Low 400 ** 300 200 * 100 (0.94) Sirius Red Fast Green 0 Nppb 16 ** 12 * RT-qPCR fold change 8 (0.45) 4 FP WGA 0 n=3 5 4 5 AAV-Cre: High High Midw Lo %FP+ CM: 77% 46% 11% Actn2: F/+ F/F High Exposure EFACTN2+ ACTN2- DEVELOPMENTAL BIOLOGY ACTN2 ACTN2 TOM ACTN2ΔE2-6 100 ; F/F

tdTomato 80

Rosa 60

ACTN2 ACTN2 GFP ACTN2ΔE2-6 40 ; Actn2

F/F 20 Isolated CM(%) Cas9GFP

Actn2 0 Rosa G- G+ T+T- FP= Cas9GFP Tomato

Fig. 1. Characterization of mice with mosaic Actn2 mutation. (A) Survival curve of Actn2F/F;RosaFP/FP mice treated with low, mid, high, and full doses of AAV- Cre. (B) Effect of AAV-Cre dosage on heart function and chamber size at P30. Left ventricle (LV) function and size were assessed echocardiographically. FS, LV fractional shortening; LVID;s, LV internal diameter at end systole; LVID;d, LV internal diameter at end diastole. (C) Effect of AAV-Cre dosage on myocardial fibrosis. P30 heart sections were stained with sirius red/fast green (Top; scale bar, 200 μm) or wheat germ agglutinin (WGA, Bottom; scale bar, 20 μm). The fraction of FP+ cells is indicated below images. (D) Real-time qPCR (RTq-PCR) analysis of Nppa and Nppb in P30 heart ventricles. (E) Representative images of ACTN2 immunofluorescence in P30 FP+ and FP− cardiomyocytes that were isolated from the same heart. The arrows point to ACTN2 mutant cardiomyocytes. The boxed area is enlarged and overexposed to show the presence of mutant ACTN2 protein (scale bar, 20 μm). (F) Quantification of cardiomyocytes with decreased ACTN2 signal (ACTN2−) among FP+ and FP− cardiomyocytes. G, Cas9GFP; T, tdTomato. Error bar, SD. n = 3 hearts per group. Box plots are described in Materials and Methods. Two-tailed Student’s t test versus control: *P < 0.05, **P < 0.01, ***P < 0.001. Parentheses indicate nonsignificant P values.

unprecedented opportunity to perform loss-of-function analysis of sarcomere filaments were misaligned, and M-lines, an ultrastruc- the cell-autonomous roles of ACTN2-based sarcomere organization tural hallmark of mature sarcomeres (16), were not detected in in vivo. mutant cardiomyocytes (Fig. 2C, yellow arrows). We next investigated the impact of Actn2 mutation on the Impact of Actn2 on Morphological Maturation. Because a major maturation of other ultrastructures. T-tubules were imaged by function of ACTN2 is to cross-link F-actin, we first examined the confocal optical sectioning of hearts perfused with the plasma impact of Actn2 mutation on F-actin organization in car- membrane dye FM 4-64 (15). FP+ mutant cardiomyocytes diomyocytes. Compared to ACTN2+ cardiomyocytes, ACTN2− exhibited reduced T-tubule content as quantified by AutoTT mutant cardiomyocytes exhibited drastically reduced F-actin as software (17) (Fig. 2 D and E). Residual T-tubules in mutant measured by phalloidin fluorescence intensity (Fig. 2 A and B). cardiomyocytes appeared disorganized and showed greater sig- Residual F-actin was retained in the mutant cardiomyocytes at the nal intensity than control cardiomyocytes (Fig. 2D). FACS–EM cell periphery, while a small number of short F-actin fragments analysis revealed dramatic dilatation of residual T-tubules in (Fig. 2A, arrows) could be observed in the cell interior, suggesting mutant cardiomyocytes (Fig. 2F, yellow arrows) adjacent to the breakdown. To further investigate the impact of remnant sarcomere Z-lines (Fig. 2F, red arrows). Actn2 mutation on sarcomere structures, we performed electron During cardiomyocyte maturation, mitochondria greatly expand microscopy (EM) on cardiomyocytes that were purified by FACS and adopt a highly organized pattern adjacent to sarcomeres. To (FACS–EM) (3, 15). Z-lines, the electron-dense boundaries of study the effect of Actn2 mutation on structural maturation of sarcomeres (Fig. 2C, red arrows), were dramatically diminished in mitochondria, we performed optical confocal sectioning of hearts the remnant myofibrils in mutant cardiomyocytes. Longitudinal perfused with the mitochondrial dye tetramethylrhodamine methyl

Guo et al. PNAS | 3of11 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling https://doi.org/10.1073/pnas.2008861118 Downloaded by guest on October 4, 2021 ABC

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Fig. 2. The impact of Actn2 mutation on cardiomyocyte morphological maturation. Mosaic mutation of Actn2 was achieved by low-dose AAV-Cre treatment of Actn2F/F;RosaFP/FP mice. Control cells were either FP− or ACTN2+ cells from the same heart (A, D, G, J, and K) or FP+ cardiomyocytes from Actn2F/+;RosaFP/FP mice (C, F, and H). Mice were treated on P1 and analyzed at P30. In A, D,andG, orange boxed areas are enlarged to the right. (A) Phalloidin staining on control and Actn2 mutant cardiomyocytes. The arrows point to small F-actin fragments. (B) Quantification of fluorescence intensity of phalloidin staining. (C) EM analysis of sarcomere ultrastructure in FACS-sorted Tomato+ control and mutant cardiomyocytes. Red arrows, Z-lines. Yellow arrows, M-lines. (D) Confocal optical sections of hearts perfused with FM 4-64, a dye that labels the plasma membrane. The stained membranes within cardiomyocytes are T-tubules. (E) T-tubule quantification by AutoTT software. (F) EM analysis of T-tubules in FACS-sorted Tomato+ control and mutant cardiomyocytes. Red arrows, Z-lines. Orange arrows, T-tubules. (G) Confocal optical sections of hearts perfused with mitochondrial dye TMRM. Orange boxed areas are enlarged to the right. (H) EM analysis of mitochondria. (I) Quantification of mitochondrial area and density by EM. (J) Cardiomyocyte geometry, measured on dissociated car- diomyocytes. (K) Representative traces (Left) and quantification (Right) of contractile cardiomyocytes upon electrical stimulation. The error bar is SD. n = 3 hearts per group. (Scale bars, 20 μmforA, D, and G or 500 nM for C, F, and H.) Box plots are described in Materials and Methods. Two-tailed Student’s t test versus control: *P < 0.05, **P < 0.01, ***P < 0.001.

ester (TMRM). TMRM signal intensity was retained in Actn2 of immunostained cardiomyocytes isolated from AAV-Cre–treated F/F FP/FP mutants, indicating preserved mitochondrial membrane potential, Actn2 ;Rosa hearts. Mutant ACTN2− cardiomyocytes exhibi- but the spatial distribution of mitochondria was perturbed (Fig. 2G ted decreased cell area due to a reduction of both cell length and and SI Appendix,Fig.S4). FACS–EM analysis revealed a decreased width (Fig. 3J). The length-to-width ratio also decreased, indi- mitochondrial cross-sectional area in Actn2 mutants, although mi- cating that ACTN2 plays a more profound role in cardiomyocyte tochondrial density did not change (Fig. 2 H and I). elongation than widening. We electrically stimulated Actn2 mu- Enlarged size and elongated shape are other key morphological tant and control cardiomyocytes and found that cardiomyocyte F/F hallmarks of cardiomyocyte maturation. We measured the geometry contraction was completely abolished in ∼80% of FP+ Actn2

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Fig. 3. Cell-autonomous impact of Actn2 on transcription. (A) Experimental design to evaluate cell-autonomous regulation of the cardiomyocyte tran- scriptome downstream of Actn2. Low-dose AAV-Cre was administered to P1 mice. Transduced cardiomyocytes were isolated at P14, FACS purified, and analyzed by RNA-Seq. Control and mutant mice had the indicated genotypes. (B) M (log ratio)-versus-A (average) plot (MA plot) of RNA-Seq results. n = 5 per group. (C) Myh6/Myh7 and Tnni3/Tnni1 reads per kilobase million (RPKM) ratios in control versus mutant groups by RNA-Seq. (D) Differentially expressed genes in Actn2 mutation are compared to Srf KO. (Left) Overlap of significantly up- and down-regulated genes is depicted. (Right) Correlation analysis of the fold change of shared differentially expressed genes between Actn2 mutation and Srf KO is depicted. (E) Top GO terms are enriched among genes down-regulated in Actn2 mutation and Srf KO experiments. Negative scores mean down-regulation. (F) Enrichment of top up- and down-regulated genes in Actn2 mutation among genes with the most significant up- or down-regulation in Srf KO. The analysis was performed using GSEA. (G) Correlation of expression change of key sarcomere, electrophysiology/Ca2+ handling, and mitochondrial maturation genes between Actn2 mutation and Srf KO experiments. r, Pearson correlation coefficient.

cardiomyocytes (Fig. 2K), consistent with the frequency of FP+ beyond its canonical role as a structural protein. To test this cardiomyocytes with mutant ACTN2 (Fig. 1F). hypothesis, we purified control and mutant cardiomyocytes by flow Together, these data indicate that ACTN2 is critical for the cytometry at P14 and performed RNA-Seq using an established establishment of specialized cardiomyocyte ultrastructure and protocol (3) (FACS–RNA-Seq, Fig. 2A and Dataset S1 and SI morphology during maturation. Appendix,TableS1). Five biological replicates for each group were SI Ap- Actn2 clearly separated by principal component analysis (PCA, Impact of on Transcription through MRTF-SRF Signaling. pendix E Transcriptional regulation is essential to coordinate the diverse ,Fig.S3 ). Differential expression analysis identified 1,352 hallmarks of cardiomyocyte maturation. The wide spectrum of or 1,287 genes that were up- or down-regulated in mutant car- phenotypes in Actn2 mutant cardiomyocytes led us to hypothe- diomyocytes (Fig. 3B and Dataset S1). Sarcomeric gene isoform size that ACTN2 regulates signal transduction and transcription switching from Myh7 to Myh6 and from Tnni1 to Tnni3 are well-

Guo et al. PNAS | 5of11 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling https://doi.org/10.1073/pnas.2008861118 Downloaded by guest on October 4, 2021 established hallmarks of transcriptional maturation in murine both fluorescence imaging and real-time qPCR, SRF depletion cardiomyocytes. RNA-Seq demonstrated a drastic decrease of significantly reduced mCherry expression (SI Appendix, Fig. S6 E Myh6/Myh7 and Tnni3/Tnni1 ratios in Actn2 mutants (Fig. 3C), and F). These data indicate that Tpm1 promoter activity is de- which supported the role of Actn2 in transcriptional maturation. pendent on SRF, suggesting that Tpm1 regulation downstream To better interpret the defects in Actn2 mutants at the tran- of ACTN2 indeed depends on SRF. scriptomic level, we purified wild-type P6 and P30 cardiomyocytes We hypothesized that Actn2 regulates gene transcription in and performed RNA-Seq analysis to yield a reference dataset for cardiomyocyte maturation through the actin-MRTF-SRF sig- the normal transcriptional changes between these two time points naling axis. To test this hypothesis, we first analyzed the car- (SI Appendix,Fig.S5A and Table S1 and Dataset S1). P6 and P30 diomyocyte RNA-Seq data to compare the transcript level of all samples were well separated by PCA (SI Appendix,Fig.S5B). Over six actin genes. A single isoform, Actc1, was responsible for 3,000 genes were up- or down-regulated from P6 to P30 (SI Ap- ∼96.0% of total actin mRNA in cardiomyocytes (Fig. 4A). We F/+ F/F pendix,Fig.S5C and Dataset S1). Cell division and DNA repli- next injected high-dose AAV-Cre into Actn2 and Actn2 cation were identified as the major biological processes that were pups at P1 and collected heart tissue at P7 for biochemical actin down-regulated (SI Appendix,Fig.S5D), consistent with the exit of analysis. This approach circumvented perturbation of the actin cardiomyocytes from the cell cycle during maturation. Tricarbox- during cardiomyocyte isolation and FACS purifi- ylic acid cycle and (GO) terms cation. Although Actn2 mutation strongly reduced F-actin were up-regulated (SI Appendix,Fig.S5D), agreeing with the in- (Fig. 2 A and B), total actin (panActin) and ACTC1 protein crease of oxidative respiration and contractile functions during levels were not changed (Fig. 4B). The decrease of F-actin and cardiomyocyte maturation. Gene set enrichment analysis (GSEA) lack of change of total actin or the predominant actin isoform (18) showed that the down-regulated genes in Actn2 mutants were ACTC1 suggested that Actn2 mutation increased G-actin. greatly enriched among the genes up-regulated during maturation, To directly measure G-actin levels, we adopted an actin pel- while up-regulated genes in Actn2 mutants were enriched for leting assay (19), in which ultracentrifugation pelleted F-actin genes down-regulated during maturation (SI Appendix,Fig.S5E). from heart lysates while leaving G-actin in the supernatant. As This result strongly indicates that Actn2 mutant cardiomyocytes a positive control, we treated heart lysates with Swinholide A, a are transcriptomically immature. small molecule that severs F-actin and triggers its depolymer- SRF is a key transcription factor regulating cardiomyocyte ization (20). This treatment significantly increased the amount of maturation (3). Therefore, we next compared Actn2 mutant and G-actin in the supernatant (SI Appendix, Fig. S7 A and B), val- Srf knockout (KO) RNA-Seq data (3), which were produced at the idating this assay for analysis of actin polymeric states in heart same age using the same experimental and analytical protocols. tissue. The actin pelleting assay showed that most ACTC1 pro- Differentially expressed genes in these datasets were highly con- teins were assembled into F-actin in control heart tissues cordant: 60.0% of down-regulated and 57.1% of up-regulated (Fig. 4C and SI Appendix, Fig. S7C). Actn2 mutation significantly genes in Srf KO cardiomyocytes were differentially expressed in increased the fraction of ACTC1 G-actin by 1.7-fold (Fig. 4C). the same direction in Actn2 mutant cardiomyocytes (Fig. 3D). In other cell types, G-actin binds MRTF to reduce its nuclear Moreover, the relative changes in expression of these shared dif- localization. Therefore, we assessed the impact of Actn2 mutation on ferentially expressed genes were highly correlated (Pearson’s r = MRTF localization. Previous studies reported conflicting results 0.93) (Fig. 3D). GSEA (18) showed that the top 10 down- about MRTF localization in cardiomyocytes (21, 22), likely due to its regulated GO terms, which were related to metabolism, mito- low protein levels in the heart and limited signal-to-noise ratio of chondria, and translation, were identical in the two experiments available antibody staining. To circumvent these problems, we gen- (Fig. 3E). The top 250 down- or up-regulated genes in Actn2 erated an AAV vector expressing MRTFA tagged with GFP. We mutants were highly enriched among the most down- or up- transduced wild-type hearts with AAV-MRTFA-GFP, perfused the regulated genes in Srf KO, respectively (Fig. 3F). We further heart with Hoechst dye to label nuclei, and then imaged in situ by performed correlation analysis using three panels of genes critical confocal optical sectioning. This method avoided potential artifacts for mature cardiomyocyte sarcomeres, electrophysiology and Ca2+ arising from tissue processing, cardiomyocyte isolation, or nonspe- handling, and mitochondria (Fig. 3G). Across these gene panels, cific antibodies and clearly revealed predominant nuclear localiza- there was excellent correlation (r > 0.6) between Actn2 mutant tion of MRTFA at P7, P14, and P30 (SI Appendix,Fig.S8A). We and Srf KO experiments. These data suggest that Actn2 and Srf also stained wild-type dissociated cardiomyocytes with a widely used function in the same pathway to regulate gene expression. MRTFA antibody (23) and observed similar nuclear localization of We previously mapped cis-regulatory elements that were endogenous MRTFA (SI Appendix,Fig.S8B). Antibody staining of bound by endogenous SRF protein in maturing murine hearts by dissociated cardiomyocytes transduced by AAV-MRTFA-GFP biotin-mediated chromatin precipitation followed by sequencing dramatically increased nuclear staining compared to nontransduced (3). Based on these data, we found that ∼53% of down-regulated cardiomyocytes (SI Appendix,Fig.S8B), further authenticating this genes in Actn2 mutants were associated with SRF-bound pro- immunolocalization. Morphological analysis of MRTFA-GFP moters (within 1 kb of transcriptional start sites; SI Appendix, overexpressing cardiomyocytes demonstrated T-tubule disorganiza- Fig. S6A). To functionally validate the impact of SRF on these tion and perturbed Z-line spacing (SI Appendix,Fig.S8C–E), promoters in vivo, we took the promoter of the actin filament consistent with deleterious effects of both increased and decreased binding protein 1 (Tpm1) as an example. The Tpm1 MRTF-SRF activity on cardiomyocyte maturation (3). F/F Cas9GFP/Cas9GFP promoter is strongly bound by SRF in maturing P14 car- Next we treated Actn2 ;Rosa mice with low- diomyocytes (SI Appendix, Fig. S6B). We designed an AAV re- dose AAV-Cre at P1 and immunostained cardiomyocytes iso- porter assay in which the Tpm1 promoter was positioned lated from P30 hearts with the MRTFA antibody. The control and upstream of mCherry (SI Appendix, Fig. S6C). To make this mutant cardiomyocytes were isolated from the same heart, stained reporter compatible with Cre-mediated mosaic gene inactiva- in the same dish, and imaged in parallel using the same confocal tion, we inserted a loxP-stop-loxP cassette between the promoter parameters. The control and mutant cardiomyocytes were distin- and mCherry so that mCherry could be driven by the Tpm1 guished by GFP signal, and MRTFA nuclear and cytoplasmic promoter only in Cre-expressing cells (SI Appendix, Fig. S6C). signals were quantified by fluorescence intensity. Quantification of We first validated by fluorescence imaging that the mCherry the nuclear-to-cytoplasmic MRTFA signal ratio showed that it was expression was indeed dependent on codelivery of AAV-Cre (SI significantly lower in Actn2 mutant cardiomyocytes (Fig. 4D). Appendix, Fig. S6D). Next, we coinjected AAV-Cre and the To validate this result using an orthogonal method that cir- Tpm1-mCherry reporter into SrfF/+ and SrfF/F littermates. By cumvented potential confounding effects of cardiomyocyte isolation,

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Fig. 4. Impact of Actn2 mutation on actin polymerization and MRTF localization. (A) mRNA expression of all six actin isoforms in control and Actn2 mutant cardiomyocytes by RNA-Seq. (B) Western blot analysis of actin in P7 heart tissues treated with high-dose AAV-Cre at P1. (C) Western blot (Top) and densi- tometric quantification (Bottom) of F-actin:G-actin ratio by actin pelleting analysis. The error bar is SD. High-dose AAV-Cre was injected at P1, and the heart tissues were collected at P7. (D) MRTFA localization in cardiomyocytes isolated from hearts with mosaic Actn2 mutation. Mosaic dose AAV-Cre was given at P1, and cells were analyzed at P30. Cells were immunostained with an MRTFA antibody and imaged using a confocal microscope (scale bar, 20 μm). (Right) quantification of fluorescence intensity ratio between nuclear and cytoplasmic MRTFA signals. (E) Western blot analysis of MRTFA, LMNB1 (nuclear marker), and GAPDH (cytoplasmic marker) in nuclear (N) and cytoplasmic (C) fractions. High-dose AAV-Cre was injected at P1, and the heart tissues were collected at P7. (Right) Quantification of MRTFA Western blot signal in the cytoplasmic fraction, normalized to GAPDH. (F) Immunofluorescence images of NIH 3T3 cells expressing FLAG-MRTFA and HA-ACTC1R64D (scale bar, 20 μm). ACTC1R64D is an ACTC1 mutant that is expected to preferentially acquire the G-actin state. (G) Quantification of N and C MRTFA fluorescence intensity. MRTFA-ΔN is a mutant MRTFA in which an N-terminal fragment containing actin-binding motifs is deleted. (H) Reciprocal coimmunoprecipitation analysis of MRTFA and ACTC1 interaction in human embryonic kidney cells 293t (HEK293T) cells. The control is an empty plasmid that does not express FLAG- or HA-tagged . IP, immunoprecipitation; IB, immunoblotting. (I) Model for ACTN2-MRTF-SRF sarcomere-to-nucleus signaling. ACTN2 promotes F-actin assembly into sarcomeres and decreases G-actin level, which allows MRTF to enter the nucleus and stimulate transcription through SRF. Box plots are described in Materials and Methods. Two-tailed Student’s t test versus control: *P < 0.05, ***P < 0.001.

F/F Cas9GFP/Cas9GFP we performed subcellular fractionation of Actn2 ;Rosa expected subcellular fractions (Fig. 4E), confirming clean sep- F/+ Cas9GFP/Cas9GFP and Actn2 ;Rosa ventricles that were treated with aration of these compartments. MRTFA was enriched in the high-dose AAV-Cre at P1 and collected at P7. Nuclear and cy- nuclear fraction, and quantification showed that cytoplasmic toplasmic markers LMNB1 and GAPDH were found in the MRTFA was greater in Actn2 mutant hearts as compared to

Guo et al. PNAS | 7of11 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling https://doi.org/10.1073/pnas.2008861118 Downloaded by guest on October 4, 2021 controls (Fig. 4E). This observed change in cytoplasmic MRTFA Perturbation of Cardiomyocyte Maturation by MRTF Inhibition. To likely under-represents the actual change because of the incomplete directly test the role of MRTFs in cardiomyocyte maturation, we depletion of ACTN2 and the diluting effect of MRTFA in non- used low-dose AAV to express a dominant-negative mutant of myocytes and nontransduced cardiomyocytes in the tissues. MRTFA (dnMRTFA) (25). This MRTFA variant lacks the We further tested whether ACTC1 interacts with MRTFA and transactivation domain (TAD, Fig. 5A) and competes with en- regulates in nuclear localization. An arginine-to-aspartate mu- dogenous MRTFA and MRTFB for interaction with SRF. As a tation at the 62nd residue of β-actin (ACTBR62D) has been result, this approach inhibits both MRTF isoforms. dnMRTFA- widely used to mimic the G-actin state of β-actin (24). This GFP was found mainly in the cell nucleus (Fig. 5B), confirming residue, corresponding to the 64th residue of ACTC1, and that the low G-actin level in wild-type cardiomyocytes favors neighboring amino acids are 100% conserved in ACTC1. We nuclear localization of MRTFA as well as dnMRTFA-GFP. We – expressed MRTFA in NIH 3T3 cells and observed nuclear observed severe T-tubule defects in dnMRTFA-GFP expressing B C MRTFA localization. However, coexpression of MRTFA with cardiomyocytes (Fig. 5 and ). These cells also exhibited R64D perturbed ACTN2 organization, characterized by the presence of ACTC1 was sufficient to reduce MRTFA nuclear localiza- D tion (Fig. 4F). In contrast, ACTC1R64D was not able to perturb longitudinal ACTN2 staining (Fig. 5 ) and decreased distance Δ E the nuclear localization of MRTFA N, a mutant lacking between Z-lines (Fig. 5 ). Maturational cardiomyocyte hyper- trophy was also perturbed by dnMRTFA (Fig. 5F). We further MRTFA’s canonical actin-binding domains (Fig. 4G). The in- R64D confirmed these phenotypes by mosaic inactivation of both Mrtfa teraction between MRTFA actin-binding domains and ACTC1 −/− F/F Cas9GFP and Mrtfb. Mrtfa ; Mrtfb ; Rosa26 were treated with a was further validated by reciprocal coprecipitation analysis mosaic dose of AAV-Cre. The Mrtfa/Mrtfb double mutant car- (Fig. 4H). diomyocytes exhibited similar T-tubule and Z-line spacing phe- We wondered if perturbation of other genes involved in sar- notypes (SI Appendix, Fig. S10 A–E). These morphological comere assembly would similarly disrupt ACTC1-MRTF-SRF phenotypes caused by MRTF inhibition were almost identical to signaling and impair cardiomyocyte maturation. Therefore, we observations in Srf KO cardiomyocytes (3), indicating a key role used CASAAV to achieve mosaic inactivation of the actin fila- of MRTFs in the activation of SRF activity, which is essential for ment binding protein TPM1. Cardiomyocytes with TPM1 de- the organization of ACTN2 and for proper cardiomyocyte pletion exhibited defective T-tubule maturation, F-actin/ACTN2 maturation. SI Appendix organization, and MRTFA nuclear localization ( , We next performed FACS–RNA-Seq to characterize the im- A–G Fig. S9 ). Thus, TPM1 and likely other genes required for pact of low-dose AAV-dnMRTFA-GFP on transcription using sarcomere assembly also regulate the ACTC1-MRTF-SRF axis. AAV-GFP–treated cardiomyocytes as control (Fig. 6A and SI Together, these data indicate that, in maturing car- Appendix, Table S1 and Dataset S1). Five biological replicates of diomyocytes, ACTN2 promotes ACTC1 polymerization and as- control versus treated cardiomyocytes were well separated by sembly into sarcomeres, which enhances nuclear localization of PCA (SI Appendix, Fig. S10F and Table S1). Statistical analysis MRTF and MRTF-SRF transcriptional activity (Fig. 4I). High revealed 2,434 down-regulated genes and 2,211 up-regulated Actn2 expression (SI Appendix, Fig. S9H), low G-actin levels (SI genes (Fig. 6B and Dataset S1). A total of 33% of genes dif- Appendix, Fig. S7C), and nuclear MRTF localization (SI Ap- ferentially expressed in dnMRTFA treatment and Actn2 muta- pendix, Fig. S8A) were observed throughout cardiomyocyte tion overlapped (Fig. 6C), and the relative expression change of maturation. Collectively, these findings suggest that ACTC1-MRTF- these shared differentially expressed genes were highly corre- SRF signaling is continuously active to promote cardiomyocyte lated (r = 0.88; Fig. 6D). The down-regulated genes were maturation in vivo. enriched for the same top-ranked GO terms, as assessed by

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Fig. 5. Perturbation of morphological maturation by dnMRTFA. (A) Design of dnMRTFA-GFP overexpression experiments. RPEL, actin-binding domain. (B) Basic region. QR, -rich region; SAP, SAP domain; LZ, zipper domain. (B) In situ confocal optical sectioning of hearts with mosaic dnMRTFA- GFP expression. Hearts were perfused with Hoeschst dye and FM4-64 (scale bar, 20 μm). The arrows point to treated cells with defective T-tubule organization. (C) AutoTT quantification of T-tubule content of dnMRTFA-GFP expressing and nonexpressing cardiomyocytes. (D) Immunofluorescent staining of ACTN2 in isolated cardiomyocytes. (E) Quantification of Z-line distance. (F) Size and aspect ratio of dnMRTFA-GFP and control cardiomyocytes that were isolated from hearts with mosaic dnMRTFA expression. Box plots are described in Materials and Methods. Two-tailed Student’s t test versus control: *P < 0.05, **P < 0.01, ***P < 0.001.

8of11 | PNAS Guo et al. https://doi.org/10.1073/pnas.2008861118 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling Downloaded by guest on October 4, 2021 GSEA (Fig. 6E). This correlation was further validated by GSEA cardiac actin isoform, ACTC1, was not previously connected to analysis of dnMRTFA data using custom gene sets containing the MRTFs. Here, we show that sarcomeres use actin-MRTF-SRF topup-anddown-regulatedgenesinActn2 mutants (Fig. 6F). signaling to modulate SRF activity during cardiomyocyte matu- These data demonstrate similar impacts of Actn2 mutation and ration. Together with the knowledge that SRF signaling also MRTF inhibition on transcription in cardiomyocyte maturation activates sarcomere assembly (3), these two signaling axes po- and support the hypothesis that they function in the same tentially build a positive feedback loop to ensure robust and signaling pathway. coordinated maturation of both cardiomyocyte ultrastructure and transcription (Fig. 6G). Discussion Actn2 mutation resulted in additional transcriptional dysregu- Sarcomere Z-lines function as signal transduction hubs to lation that did not overlap with Srf KO or dnMRTF treatments modulate cardiac disease pathogenesis (5), but whether sarco- (Figs. 3D and 6C), suggesting the regulation of other molecular meres regulate signaling during heart development was unclear. pathways. For example, Actn2 could potentially regulate CSRP3 Manipulation of sarcomere components in animals often impacts (also known as MLP), which was reported to shuttle between overall heart function and consequently results in secondary ef- Z-lines and nucleus (26) to maintain normal cardiac function (27). fects that complicate the analysis of sarcomere functions. In this Alternatively, sarcomere disassembly could impair cell contrac- study, we combined AAV-mediated genetic mosaic analysis (9) with a newly generated floxed allele of Actn2 to efficiently dis- tility and induce transcriptional changes through other mecha- assemble sarcomeres in cardiomyocytes in vivo while maintaining notransduction pathways. Future studies are required to further overall normal cardiac function. This approach revealed a cell- dissect the mechanisms that govern ACTN2-dependent tran- autonomous role for ACTN2 in the ultrastructural maturation of scriptional regulation. cardiomyocytes, which was coupled with a profound impact on Cardiac pathological remodeling is characterized by changes gene transcription. that overlap with cardiomyocyte maturation defects in several We identified at least one molecular mechanism that con- aspects. One representative change is the reactivation of a fetal- tributes to this sarcomeric regulation of transcription: sarcomere like transcription program. By performing mosaic analysis, we assembly activates MRTF-SRF signaling by promoting ACTC1 excluded the interference of noncell autonomous pathological polymerization. Although the importance of actin-MRTF-SRF changes on our study of cardiomyocyte maturation. However, we signaling is established in noncardiomyocytes, it was unknown if recognize that genetic mosaics cannot exclude cell-autonomous

this mechanism was active in cardiomyocytes. The actin cyto- activation of pathological stress responses. Identification of such DEVELOPMENTAL BIOLOGY skeleton of cardiomyocytes is highly specialized, and the major stress responses and their impact on cardiomyocyte maturation

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Fig. 6. Transcriptomic perturbation by dnMRTFA. (A) Design of RNA-Seq analysis of dnMRTFA treatment. Mice were treated with low-dose AAV-dnMRTFA-GFP or AAV-GFP. GFP+ cardiomyocytes were purified by FACS and analyzed by RNA-Seq. (B) MA plot of gene expression in dnMRTFA-GFP compared to GFP cardiomyocytes. n = 5 per group. (C) Venn diagram comparing genes differentially expressed in Actn2 mutation or dnMRTFA expression. (D) Correlation analysis of genes differentially expressed genes in both Actn2 mutation and dnMRTFA expression experiments. r, Pearson correlation coefficient. (E) The 10 GO terms most enriched among genes down-regulated in dnMRTFA-treated or Actn2 mutant cardiomyocytes were the same. (F) The top 250 up- or down-regulated genes in Actn2 mutants were highly enriched among the up- or down-regulated genes, respectively, in dnMRTFA-expressing cardiomyocytes. The analysis was performed using GSEA. (G) Model of mutual potentiation between sarcomere assembly and MRTF-SRF signaling to coordinate cardiomyocyte maturation.

Guo et al. PNAS | 9of11 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling https://doi.org/10.1073/pnas.2008861118 Downloaded by guest on October 4, 2021 would be critical to more precisely define the mechanisms that CASAAV. The protocols to perform CASAAV experiments were previously promote or perturb cardiomyocyte maturation. described (8, 10). The gRNA sequences in this study are as follows: Actn2,5′- ACTN2 GATGGTCCAGATCATACCCA-3′,5′-TCGCCCACAGAAATAGTCGA-3′,5′-GAC- Human sarcomere gene mutations, including muta- ′ ′ ′ ′ tions, are well-known causes of inherited cardiomyopathies, such ATAGTTGTACTGCACGC-3 , and 5 -CCGCGACCTGCTCCTCGACC-3 ; Tpm1,5- TCTGAACAGACGCATCCAGC-3′ and 5′-AGGCTGCAGATGAGAGTGAG-3′. as dilated cardiomyopathy and hypertrophic cardiomyopathy. While conventional mechanistic studies of these diseases have In Situ Heart Optical Confocal Sectioning. In situ cardiac imaging was per- focused on sarcomere contractile function or calcium sensitivity, formed as previously described (15). Hearts were perfused with FM 4-64 the discovery of ACTN2-ACTC1-MRTF-SRF signaling suggests (Invitrogen, 13320), TMRM (Life Technologies), or Hoechst 33342 (Invi- altered signal transduction could contribute to disease patho- trogen) dyes as indicated. The heart was then positioned on a glass-bottom genesis caused by a subset of sarcomere mutations. This hy- dish and immediately imaged using an Olympus FV3000RS inverted confocal pothesized mechanism also predicts that disruption of the microscope. coordinated cardiomyocyte maturation program could contrib- – ute to cardiac disease pathogenesis. Consistent with this idea, a FACS EM. Isolated cardiomyocytes in suspension were fixed with 4% para- formaldehyde for 30 min at room temperature. The fixed cells were sorted on recent study demonstrated cardiomyocyte maturation defects a BD AriaII SORP cell sorter with a 100-μm nozzle. After FACS, the cells were and congenital heart malformation as a consequence of disease- fixed again in 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M of So- causing mutations in MRTFB, MYH7 (a major component of dium Cacodylate buffer, pH 7.4, overnight at 4 °C. The cell pellets were next mature sarcomere thick filaments in humans), and an additional processed for routine transmission EM protocol. genetic modifier (28). Cardiomyocytes that are derived from nonmyocytes or stem RNA-Seq and Data Analysis. Total RNA from FACS-sorted cardiomyocytes was cells are widely used in disease modeling, pharmacological tests, reverse transcribed, and full-length complementary DNA was specifically tissue engineering, and cell therapy trials. These cells exhibit amplified for eight PCR cycles using SMART-Seq v4 Ultra Low Input RNA Kit immature phenotypes in vitro, imposing a major bottleneck on (Clontech). DESeq2 (32) was used to perform statistical analysis of differen- tial gene expression, with differentially expressed genes defined by adjusted cardiac regenerative medicine (29). Many studies have used P < 0.05. An adjusted P value of 0.05 was used as the cutoff to identify ACTN2 staining patterns as a key hallmark of in vitro car- differentially regulated genes. diomyocyte maturation. Here, we provide an insight that im- proved ACTN2 organization mechanistically contributes to Statistical Analysis. Statistical analysis and plotting were performed using JMP cardiomyocyte maturation and is not merely a quantifiable end software (SAS Institute). Statistical tests are indicated in each figure legend. point. We propose that sarcomere maturation is a key prereq- The numbers in parentheses in figures indicate nonsignificant P values. The uisite to overall cardiomyocyte maturation. bar plots show mean ± SD. In the box plots, horizontal lines indicate the median and 25th and 75th quantiles; whiskers extend 1.5 times the inter- Materials and Methods quartile range from the 25th and 75th percentiles; and dots represent possible outliers. Please see SI Appendix, Supplemental Materials and Methods for additional information. Data Availability. RNA-Seq data generated for this study have been deposited in the Gene Expression Omnibus database under the accession code Mice. All animal strains and procedures were approved by the Institutional F GSE136096. Actn2 mice, produced in Dr. Alan Beggs’s laboratory, can be ’ F Animal Care and Use Committee of Boston Children s Hospital. Actn2 mice obtained through a material transfer agreement. were generated by homologous recombination in embryonic stem cells. RosaCas9GFP/Cas9GFP (13) and RosaTomato/Tomato (14) were imported from the −/− F/F ACKNOWLEDGMENTS. We appreciate the technical support by Dana-Farber Jackson Laboratory. Mrtfa (30) and Mrtfb (31) mice were reported Flow Cytometry Core, Harvard Medical School (HMS) Electron Microscopy previously. Core, HMS Biopolymers Facility, and Boston Children’s Hospital (BCH) Animal Facility. We thank Dr. Guido Posern for sharing the MRTFA anti- AAV. AAV9 was prepared as previously described (3, 8). AAV titer was body. This project was supported by NIH (R01HL146634 and UM1HL098166 to quantified by real-time PCR using a fragment of the cardiac W.T.P., R01AR044345 to A.H.B, and K99HL148309-01A1 to Y.G.), the American Heart Association (postdoctoral fellowship 18POST33960037 to Y.G.), and char- promoter DNA to make a standard curve. PCR primers for AAV quantifica- ’ ′ ′ ′ itable support from the Boston Children sHospitalDepartmentofCardiology. tion were 5 -TCGGGATAAAAGCAGTCTGG-3 and 5 -CCCAAGCTATTGTGT- Mouse genotyping and phenotyping was supported by the resources of the ′ GGCCT-3 . AAV was injected into P1 pups subcutaneously. The P1 pups were Intellectual and Developmental Disabilities Research Center (IDDRC) Molecular 8 anesthetized in an isoflurane chamber before injection, and 3.6 × 10 vg/g Genetics Core funded by U54HD090255 from the National Institute of Child was used in all mosaic analyses. Health and Human Development (NICHD) of NIH.

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Guo et al. PNAS | 11 of 11 Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling https://doi.org/10.1073/pnas.2008861118 Downloaded by guest on October 4, 2021