Mouse strains. Malat1–/– and Malat1fl/fl mouse lines were described previously (38). Wild-type littermates were used as control for the Malat1–/– mice. Cardiomyocyte-specific deletion of Malat1 was established by cross-breeding Malat1fl/fl mice with a Myh6-Cre line (39). Malat1fl/fl;Myh6-Cre+/+ mice are referred to as control and Malat1fl/fl;Myh6-CreCre/+ mice are referred to as Malat1ΔCM. Cardiomyocyte-specific deletion of Malat1 was validated by RT-qPCR analysis from cardiomyocyte RNA. Mice were housed at 23°C ambient temperature and 60% humidity in 12-hour light/dark cycles.

MI. Neonatal mice on P3 were anesthetized by inducing hypothermia on ice, as described previously (2, 33). After transverse skin incision, lateral thoracotomy was applied at the fourth intercostal space by blunt dissection of intercostal muscles. To induce MI, an 8-0 prolene suture (Ethicon) was passed through the heart and was tied below the LAD coronary artery. Using a nonabsorbable 6-0 suture (Ethicon), the chest was stitched and skin glue was applied to join the skin together. Blood and remaining skin glue were removed with an alcohol solution. Pups were warmed under a heat lamp for several minutes until recovery and were placed back with their mother. Mice were euthanized 7 or 21 days after surgery.

Echocardiography. Echocardiography was performed 21 days after MI using a Vevo 3100 imaging system (VisualSonics) with the MX550 or MX700 probe. Parasternal long-axis views, short-axis views, and 2-dimensional-guided M-mode images of the short axis at the papillary muscle level were captured. To minimize variability between the measurements, all echocardiographic measurements were analyzed by VevoLab 3.2.5 software using artificial intelligence–based Auto-LV technology.

Histology. Postnatal hearts were perfused with PBS and incubated in 4% formaldehyde (Thermo Fisher Scientific, 28908) at 4°C overnight. Hearts were processed for paraffin embedding using standard procedures in the institute. Hearts were sectioned at 4 μm intervals and used for immunohistochemistry.

Sirius red staining. Slides were deparaffinized and rehydrated. Slides were stained in 0.1% Picro Sirius red solution (Waldeck and Sigma-Aldrich) according to the manufacturers’ instructions. Slides were mounted with Pertex mounting medium (Histolab, LEIC811). Images were acquired using an inverted light microscope (Nikon) with 2× objective. To determine the fibrotic score, ImageJ software (NIH, version 1.53a) was used to measure the positive fibrotic area and establish a ratio versus the total tissue area. At least 5 sections per animal were used for quantification.

TUNEL staining. TdT-mediated dUTP nick-end labeling (TUNEL) assays were conducted on paraffin-embedded heart sections. To this end, the In Situ Cell Death Detection Kit, Fluorescein (Roche, 11684795910) was used according to the manufacturer’s instructions. TUNEL-positive cells were analyzed in border, remote, and infarct zones.

Immunohistochemistry. Paraffin-embedded heart sections were deparaffinized and rehydrated by incubation in xylene followed by incubation in descending ethanol concentrations (100%, 80%, 70%, and 50% in water) and finally washed in PBS. For Aurora B staining, antigen retrieval was additionally performed. To do so, slides were boiled for 15 minutes in 10 mM citrate buffer, pH 6.0. Slides were cooled down and washed 3 times with PBS. Afterwards, sections were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, and were blocked in 2% BSA and 1% donkey serum for 1 hour at room temperature. Slides were stained with primary antibodies overnight at 4°C in a humid chamber and washed 3 times with PBS, followed by incubation with secondary antibodies for 1 hour at room temperature. Slides were mounted using Fluoromount-G mounting medium (Thermo Fisher Scientific, 00495802). Immunostainings were imaged using a Zeiss LSM780 confocal microscope and analyzed using Volocity software 6.5.1 (Quorum Technologies).

The following primary and secondary antibodies were used: rabbit anti–p-histone H3 (EMD, 06570; 1:100), mouse anti-PCM1 (Santa Cruz Biotechnology, sc398365; 1:50), rat anti-CD31 (Dianova, DIA-310; 1:30), rabbit anti–Aurora B (Abcam, ab2254; 1:500), mouse anti–troponin T (Thermo Fisher Scientific, MS295; 1:100), mouse anti-CD45 (Abcam, ab8216; 1:100), donkey anti-mouse–Alexa Fluor 555 (Invitrogen, A-31570; 1:200), donkey anti-rat–Alexa Fluor 647 (Invitrogen, A48272; 1:200), and donkey anti-rabbit–Alexa Fluor 488 (Invitrogen, A21206; 1:200).

Isolation of neonatal mouse cardiomyocytes. Cardiomyocytes were isolated from P1, P3, and P7 pups. Neonatal hearts were dissected, atria were cut off, and ventricles were collected in ice-cold 20 mM 2,3-butanedione 2-monoxime (BDM) solution in HBSS. Ventricles were then cut into 1–2 mm pieces and digested using the gentleMACS dissociation kit (Miltenyi Biotec GmBH, 130-093-235). Cells were then either plated on collagen I–coated (Thermo Fisher Scientific, A1048301) μ-Slides (Ibidi, 80826) or lysed for RNA extraction.

Immunocytochemistry. Isolated neonatal mouse cardiomyocytes or mouse HL-1 cells were cultured on μ-Slides and fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908) for 10 minutes at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature. Next, cells were blocked with 2% BSA (Sigma-Aldrich, A7030) and 1% donkey serum (Sigma-Aldrich, D9663) for 1 hour at room temperature, followed by primary antibody incubation at 4°C overnight in a humidified chamber. Cells were washed 3 times with PBS and incubated with secondary antibodies for 1 hour at room temperature. Phalloidin (Invitrogen, 1:200) was added together with the secondary antibodies to visualize the cytoskeleton. Immunostainings were imaged using a Zeiss LSM780 confocal microscope and analyzed using Volocity software 6.5.1.

The following primary and secondary antibodies were used: rabbit anti–troponin T (Abcam, ab45932; 1:1000), rabbit anti–Aurora B (Abcam, ab2254; 1:500), donkey anti-rabbit–Alexa Fluor 555 (Invitrogen, A-31572; 1:200), and donkey anti-rabbit–Alexa Fluor 555 (Invitrogen, A-31573; 1:200).

RNAScope. RNAScope (Advanced Cell Diagnostics) on paraffin-embedded heart sections was performed according to the manufacturer’s instructions. Briefly, deparaffinization and rehydration were done as described above. Sections were incubated with the probe against mouse Malat1, which spans nucleotides 712–2338 (transcript ID: NR_002847.2) or with a negative control probe (both designed by Advanced Cell Diagnostics). Probes were hybridized for 2 hours at 40°C in a hybridization oven. Signal amplification was achieved by hybridization with amplification probes serially and using tyramide signal amplification reagents provided within the kit. For immunostaining, sections were blocked with 10% donkey serum, 5% skim milk, and 0.1% Triton X-100 in PBS, followed by primary antibody incubation at 4°C overnight. Cells were washed 3 times with PBS and incubated for 1 hour at room temperature. Slides were imaged using a Zeiss LSM780 confocal laser scanning microscope equipped with 40× and 63× objectives.

The following primary and secondary antibodies were used: rabbit anti–sarcomeric α-actinin antibody (Abcam, ab68167; 1:250) and donkey anti-rabbit–Alexa Fluor 555 (Invitrogen, A-31572; 1:200).

Maintenance of mouse HL-1 cells. The murine atrial cardiomyocyte cell line HL-1 (Sigma-Aldrich, SCC065) was cultured in Claycomb medium (Sigma-Aldrich, 51800C) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 μg/mL penicillin/streptomycin (Gibco, 15140122), 0.1 mM norepinephrine (Sigma-Aldrich), and 2 mM GlutaMAX (Gibco, 35050061). Cells were cultured in cell culture dishes coated with 0.02% gelatin (Millipore) and 0.1% fibronectin from human plasma (Sigma-Aldrich, F0895) at 37°C and 5% CO2.

Gene silencing and overexpression in mouse HL-1 cells. For gene silencing experiments, cells were seeded 24 hours before transfection and were transfected with a final concentration of 50 nM LNA or 50 nM siRNA using RNAimax (Invitrogen, 13778075). Sequences of LNA-gapmers (Qiagen) and siRNAs (Sigma-Aldrich) are listed in Supplemental Table 1.

For hnRNP U overexpression experiments, wild-type and mutant hnRNP U expression plasmids were used. Mutated hnRNP U plasmid lacks the RNA binding RGG-box domain (MRGGNFRGGAPGNRGGYNRRGNMPQR). Both plasmids were designed by OriGene Technologies using the pCMV6-AC-HA mammalian expression vector as backbone (OriGene, PS100004). ORF sequences of both plasmids are listed in Supplemental Table 2. Cells were seeded 24 hours before transfection and then transfected with 2.5 μg of hnRNP U wild-type or mutant plasmid per 300,000 cells/well in 6-well plates using Lipofectamine 3000 reagent (Invitrogen, L3000001). hnRNP U overexpression efficiency was validated by RT-qPCR.

Total RNA isolation from cell culture and RT-qPCR. RNA isolation from mouse HL-1 cells was done using RNeasy mini kits (Qiagen, 74104), including an on-column DNase digestion step according to the manufacturer’s instructions. Concentration of total RNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, 1 μg of RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen, 28025013) and random hexamers (Qiagen, 79236). RT-qPCR was performed on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). Mouse ribosomal protein lateral stalk subunit P0 (Rplp0) was used for data normalization and relative expression levels were calculated by the 2–ΔCt method. Primer pairs are listed in Supplemental Table 3.

RNA sequencing. RNA was isolated as described above. Total RNA and library integrity were verified on LabChip Gx Touch 24 (PerkinElmer). Total RNA (1 μg) was used as input for SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Clontech, 634873). Sequencing was performed on the NextSeq 500 instrument (Illumina) using v2 chemistry with 1 × 75 bp single-end setup. The resulting raw reads were assessed for quality, adapter content, and duplication rates with FastQC (60). Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q20 in a window of 10 nucleotides (61). Only reads of at least 15 nucleotides were cleared for subsequent analyses. Trimmed and filtered reads were aligned versus the Ensembl mouse genome version mm10 (Ensembl release 101) using STAR 2.7.7a with the parameters “--outFilterMismatchNoverLmax 0.1 --alignIntronMax 200000” (62). The number of reads aligning to genes was counted with featureCounts 1.6.5 from the Subread package (63). Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded. Differentially expressed genes were identified using DESeq2 version 1.30.0 (64). Only genes with a minimum fold change of ±1.5 (log2 ±0.59), a maximum Benjamini-Hochberg–corrected P value of 0.05, and a minimum combined mean of 5 reads were deemed to be significantly differentially expressed. The Ensembl annotation was enriched with UniProt data (release 24.03.2017) based on Ensembl gene identifiers (activities at UniProt).

GO term analysis. GO analysis was performed using the Enrichr database (https://maayanlab.cloud/Enrichr/) (41, 42, 65).

Immunoprecipitation and Western blotting. For immunoprecipitations, mouse HL-1 cells were washed 3 times with ice-cold PBS and extracted with ice-cold RIPA buffer (Sigma-Aldrich, R0278; supplemented with protease and phosphatase inhibitors) for 30 minutes on ice. After centrifugation for 15 minutes at 4°C, the supernatant was collected and incubated with antibody-coupled protein G Dynabeads (Invitrogen, 10003D) (50 μL of beads were coupled with 6 μg antibody) overnight at 4°C. Immunoprecipitates bound to the beads were washed 3 times with wash buffer (150 mM NaCl, 0.05% NP-40, 50 mM Tris HCl pH 8.0, supplemented with protease and phosphatase inhibitors) and the proteins were eluted with Laemmli sample buffer. Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes at 100 V for 30 minutes in 1× transfer buffer (25 mM glycine, 20% methanol, 0.15% ethanolamine). Afterwards, membranes were blocked with 5% nonfat milk powder for 1 hour at room temperature. Rabbit IgG (Millipore, PP64) was used as IgG control and rabbit anti–hnRNP U antibody (Abcam, ab10297) was used for hnRNP U immunoprecipitation experiments. Antibodies for detecting hnRNP U (Abcam, ab10297; 1:1000) and GAPDH (Cell Signaling Technology, 2118; 1:2000) were diluted in 0.05% Tween 20 and 5% nonfat milk powder and incubated together with membranes overnight at 4°C. Membranes were washed 3 times with TBS-T and incubated with anti-rabbit IgG secondary antibody (Amersham, NA934v; 1:2500) for 1 hour at room temperature. After adding chemiluminescence reagent (Millipore, WBKLS0500), blots were imaged using a ChemiDoc Touch imaging system (Bio-Rad).

Statistics. All statistical analyses were done using Prism 8 (GraphPad) software (with the exception of RNA sequencing). Data are expressed as mean ± SEM. The Shapiro-Wilk normality test was used to analyze data distribution. For 2-group comparisons, statistical significance was determined using a 2-tailed Student’s t test for normally distributed samples and Mann-Whitney U test for the not normally distributed samples. For multiple comparisons, statistical significance was determined using 1-way ANOVA with Tukey’s multiple-comparison test. P values of less than 0.05 were considered significant.

Data availability. The RNA sequencing data sets generated in this study are available at the NCBI Gene Expression Omnibus database (GEO GSE200733). Source data are provided with this paper.

Study approval. All animal procedures were conducted according to the principles of laboratory animal care, institutional guidelines, and approved by local animal ethics Tierschutzbeauftragte from Goethe University Frankfurt. All animal experiments were conducted under permissions FU/1183 and FU/2035 granted by the Regierungspräsidium Darmstadt, Hessen.