Fig 2: RBPMS RBFOX2 act downstream of MYOCD. A previous study identified a putative splicing complex consisting of RBPMS, RBPMS2, and RBFOX2 (A). We noted that an additional partner in this complex (PICK1) was regulated by MYOCD at the mRNA level, suggesting that MYOCD exerts its splicing effects via 3–4 core partners (green) in a splicing factor complex. Silencing of RBPMS (B), antagonized MYOCD-driven changes in splicing of ACTN1, MBNL1, and VCL as shown using variant-specific primers and agarose gel electrophoresis (C–E, n = 6 in this and the following panels). Similarly, silencing of RBFOX2 (F), antagonized the effect of MYOCD on splicing of these transcripts (G–J). We also examined if MYOCD, but not MRTF-A, can drive differential splicing of vinculin (VCL) using western blotting (K). All MRTFs increased the meta-VCL/VCL ratio (L). RBPMS was preferentially regulated by MYOCD and MRTF-B (M), but RBPMS and the meta-VCL to VCL ratio correlated across the entire dataset (N). All MRTFs similarly promoted VCL splicing in bladder SMCs (O, numbers from the densitometric quantification are shown by the bands). Because MRTF-A and MRTF-B are inhibited by depolymerization of actin, bladder SMCs were treated with Latrunculin B, which depolymerizes actin. This reduced the mRNA levels of RBPMS and RBFOX2 (P) and inhibited SMC-specific splicing (Q, R)

Fig 3: Partial rescue of HF phenotype upon VDAC2 reintroduction.a Western blot image of VDAC2-GFP and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (KO GFP vs. KO VDAC2-GFP p = 0.0568) (n = 3); b qRT-PCR of Vdac2 normalized to Vcl (Vinculin) (KO GFP vs. KO VDAC2-GFP p = 0.0044) (n = 3); c–g Serial echocardiography parameter including ejection fraction (EF) (week 6: WT GFP vs. KO GFP p = 0.0036, week 10: WT GFP vs. KO GFP p = 0.0008, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0456, KO GFP vs. KO VDAC2-GFP p = 0.0359), fractional shortening (FS) (week 6: WT GFP vs. KO GFP p = 0.0091; week 10: WT GFP vs. KO GFP p = 0.0043, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0652), left ventricular end diastolic diameter (LVEDD) (pre: WT GFP vs. KO GFP p = 0.0169, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0407; week 10: WT GFP vs. KO GFP p = 0.0107, KO GFP vs. KO VDAC2-GFP p = 0.0444), end diastolic and end systolic volumes (EDV (pre: WT GFP vs. KO GFP p = 0.0032; week 10: WT GFP vs. KO GFP p = 0.0104, KO GFP vs. KO VDAC2-GFP p = 0.0378) and ESV (week6: WT GFP vs. KO GFP p = 0.03, week 10: WT GFP vs. KO GFP p = 0.0008, KO GFP vs. KO VDAC2-GFP p = 0.0066)) respectively (n = 3). p-value: unpaired t-test performed in all comparisons between KO GFP and KO VDAC2-GFP in panels a and b. Ordinary one-way ANOVA was performed for echocardiographic data and panel b. Data are represented as ±SEM.

Fig 4: Additional examples of MYOCD-driven differential exon usage include SLMAP, CALD1, and VCL. SLMAP which encodes sarcolemma associated protein, was among the striking examples in the exon-based analysis. Reads mapping to 3´, late, exons were increased relative to early exons (A). Late exons are represented in the two annotated mRNA isoforms shown in light blue in (B). Western blotting showed SLMAP bands at 40 and 30 kDa that increased dramatically with MYOCD compared to null virus, while bands at 80 and 70 kDa remained inert (C). After blotting for SLMAP, the membrane was stripped and re-probed for HSP90 (D). Panel E shows western blotting for caldesmon (null: −; MYOCD: +). Heavy (h-CALD1) and light caldesmon (l-CALD1) variants were resolved, and intensity was adjusted to clearly show the heavy isoform in the top blot (long), and the lower band in the middle blot (short). Quantification, shown at the bottom, demonstrated a greater relative increase of h-CALD1 compared to l-CALD1. MYOCD similarly promoted expression of meta-vinculin (F), which is a SMC-enriched ≈ 150 kDa splice variant derived from the VCL gene, relative to vinculin (≈ 130 kDa)

Fig 5: Inducible and SMC-specific knockout of Srf in vivo reduces SMC splicing of Vcl, Cald1, and Myocd. To address the in vivo relevance of Myocd-Srf-driven splicing, we generated mice allowing for inducible deletion of Srf in SMCs (Myh11-CreERT2 x Srffl/fl mice injected with tamoxifen, referred to as knockout, KO). Compared to wild type mice (WT), staining for Srf (green), which is a predominately nuclear protein, was reduced in SMCs in the aorta and urinary bladder (A). White lines in the micrographs highlight the SMC layers of the aorta and bladder, respectively. This was confirmed by RT-qPCR, showing reduction of Srf along with three contractile markers in both aorta (B), and bladder (C). We focused on the bladder in view of better Srf depletion and could confirm sizeable reduction of Srf and SMC markers by western blotting (D). Contraction in response to cumulative addition of the muscarinic agonist carbachol (E), depolarization with KCl (60 mM, F), and stimulation with the phosphatase inhibitor Calyculin A (1 µM, G), was reduced, confirming loss of contractility in KO vs. WT bladder. In addition to these expected phenotypes, we also observed a reduction of Rbpms (H), but not of Rbfox2 (I) by RT-qPCR. Splicing of Vcl, Cald1, and Mbnl1 was altered as shown using PCR and agarose gel electrophoresis (J). Myocd splicing (exon 2a) was also examined. Myocd splicing changed from the SMC variant containing exon 2a (+ Ex2a), towards a variant that lacks exon 2a (− Ex2a, panel J, bottom). Silencing of either RBPMS or RBFOX2 similarly favored the heart variant (− Ex2a) of MYOCD in cultured human coronary SMCs (K). Altered splicing of Vcl, and Cald1 in KO bladder was confirmed by western blotting (L)