Therapeutic Delivery of Pip4k2c‐Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart

Abstract Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF‐β, respectively. Type‐2‐phosphatidylinositol‐5‐phosphate‐4‐kinase‐gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non‐injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c−/− mice has higher rates of CH, CF, and sudden death than wild‐type mice. In a gain‐of‐function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGFβ1 via its N‐terminal motif, Pip5k1α, phospho‐AKT 1/2/3, and phospho‐Smad3. In sum, loss‐and‐gain‐of‐function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.


Transverse Aortic Constriction (TAC) model
All surgical and experimental procedures with mice were performed in accordance with protocols approved by IACUC and the MSSM Center for Comparative Medicine and Surgery (CCMS). Thoracic aortic constriction was performed on 8-week-old male C57BL/6J or CFW mice (Charles River Laboratories), as described previously with small adaptations 4 . Mice received buprenorphine (0.1 mg/kg s.c.) 60 min before intubation and anesthesia with isoflurane. Thoracotomy was performed between the second and third rib, and the aortic arch was narrowed by a ligature over a 27 G cannula to generate trans-stenotic pressure of approximately 40 mmHg in all animal groups. In addition to undergoing the same constriction, all mice were of the same age and body weight. Mice remained in a warmed cage for 2-4 h, until completely recovered from anesthesia, under direct supervision. In sham surgery, the chest was opened, but the aorta was not ligated. Cardiac dimensions and function were analyzed by pulse-wave Doppler echocardiography before TAC/sham surgery and before the animals were euthanized. Mice were sacrificed to determine cardiac hypertrophy, fibrosis, and other parameters. In all experiments, the surgeon was blinded to the treatment group. To assess heart histology, hearts were collected at the end of each experiment. The hearts were excised, briefly washed in PBS, perfused with perfusion buffer, weighed, and fixed in 4% PFA at 4°C overnight. The next day, hearts were washed with PBS and incubated overnight in 30% sucrose. Next, hearts were put in OCT, frozen, and stored at -80°C. The heart blocks were sectioned transversely or longitudinally at 8-9μm using a cryostat. The slides were further processed for immunostaining (see below) or histological staining with hematoxylin and eosin (H&E) and Sirius red / fast green, all performed according to standard procedures. The heart weight to tibia length ratio was measured using a standard scale at each experiment endpoint. This ratio was calculated as the heart tissue weight relative to the mouse tibia length, with weight in grams (mg) and length in mm.

Adult cardiomyocyte and non-cardiomyocyte isolation
Cardiomyocytes and non-cardiomyocytes were isolated (for Fig 1j) from adult mice hearts using Langendorff's method as previously described 60 . Briefly, hearts were excised and the aortas were cannulated and perfused with buffer A (in mM: 113 NaCl, 4.7 KCl, 0.6 KH 2 PO 4 , 0.6 Na 2 HPO4, 1.2 MgSO 4 , 12 NaHCO 3 , 10 KHCO 3 , 10 HEPES, 30 taurine). To dissociate cells, collagenase type II (Worthington) was included in the buffer. After dissociation, the cell suspension was incubated for 10 min at 37 °C, allowing the CMs to sediment. The non-cardiomyocyte-enriched supernatant and pellet were then resuspended in buffer B (47.5 ml perfusion buffer A, 2.5 ml FCS, 62.5 ml, 10 μM CaCl 2 ) and centrifuged for 5 min (400 x g). Both CM and non-CM fractions were used to isolate mRNA.

Isolating cardiac fibroblasts
Hearts were removed from TAC mice using curved forceps, minced into small pieces (<1 mm 3 in size) using scissors, and washed with cold PBS to remove any excess blood. 2ml of fibroblast explant medium (Iscove's Modified Dulbecco's Medium + 20% FBS) was added to each heart to dissociate the tissue pieces by pipetting. Next, the tissue pieces were plated evenly on 0.1% (wt/vol) gelatin-coated 6-well plates with 1/4 adult heart per well in 0.2ml of fibroblast explant medium. The tissue was allowed to attach for 2 hours, after which 2ml of additional fibroblast explant medium was added to each well. Cells were allowed to attach for 7-10 days before undergoing fibroblast sorting.

Sorting cardiac fibroblasts
Cardiac cells were isolated by explantation and cultured for up to 7-10 days till they were ready for staining and FACS. Cells were washed with PBS and dissociated with 0.05% (wt/vol) trypsin for 5 mins. An equal volume of fibroblast explant medium was added to neutralize the trypsin. Thereafter, the cells were passed through a 40-μm cell strainer to remove any tissue fragments and then concentrated by centrifugation at 600g for 5 min at RT. Next, the cells were stained with 200μl Thy-1/APC antibody (eBioscience, by diluting the antibody in 10% (vol/vol) FBS in PBS at room temperature for 30-60 mins. Cells were then washed with PBS, centrifuged at 600g for 5 min at RT, kept on ice, and sorted for Thy1+ cells. Sorted Thy1+ cells were plated at a density of 10 4 cells per cm 2 .

Total collagen assay
Isolated and sorted cardiac fibroblasts from WT or KO-pip4k2c mice 21 days post TAC operation were treated with DMSO, SB431542, Luc modRNA, Pip4k2c, or mutPip4k2c modRNA. 3 days later, cells were washed with PBS, then harvested, and lysates were evaluated for collagen production using a total collagen assay kit from Abcam (ab222942). The colorimetric signals were analyzed at OD 560 nm on a microplate reader according to standard procedures.

Echocardiography (ECHO)
We evaluated left ventricle dimensions and function by performing transthoracic twodimensional echocardiography. In a double-blind study (neither the surgeon nor the echography technician was aware of the treatment), various modRNAs were injected into C57BL/6J or CFW mice (8 to 12 weeks old). Animals underwent echography onsite with a GE Cares machine (V7R5049) equipped with a 40 MHz mouse ultrasound probe. Mice were anesthetized with a mixture of 1-2% isoflurane in air, and imaging was performed on days 2 and 28 post LAD ligation. The ejection fraction and fractional shortening were calculated as percentages from the diastolic volume (EDV) and endsystolic volume (ESV) dimensions on an M-mode ultrasound scan. The following formulas were used: % ejection fraction = (EDV-ESV)/EDV*100 and % fractional shortening = (left ventricular internal dimension at end-diastole (LVIDd)left ventricular internal dimension at end-systolic (LVIDs))/ LVIDd*100. Echocardiograms were performed on 4-10 hearts/treatment group.

Hematoxylin and eosin (H&E) staining
H&E staining was performed according to standard procedures. The OCT frozen longitudinal heart sections were dried for 30 min to 1 hr at room temperature, then hydrated in PBS for 10 mins. The slides were kept in Hematoxylin stain for 2 mins and washed with tap water for 5 mins. Next, the sections were stained using an eosin solution for 1 min and washed with tap water for 5 mins. The slides were transferred to PBS for 5 mins. Sections were then dehydrated in 100% ethanol and xylol for 1 min each. Finally, sections were mounted. The images were taken on a bright-field microscope.

Mouse survival post TAC
For our long-term survival study, the C57BL/6J or Pip4k2c -/mice (8-10 weeks old) were subjected to a survival study after either TAC operation or modRNA injection. The mice were checked every week for death or survival (Fig 2r and Fig 3q).

E18 mice study
To analyze heart development in WT or Pip4k2c -/mice, we harvested the embryonic mice at E18. We determined embryonic mouse weight and heart weight and also imaged these mice and their hearts on a bright-field microscope ( Fig S2).

Flow cytometry
Left ventricles of wild type and Pip4k2c KO mice were dissected at 21 days post TAC and processed according to a previously described protocol 61 with a few modifications.

Immunostaining heart sections
Frozen heart sections were rehydrated in PBS for 5 min, followed by permeabilization in PBS with 0.1% triton X100 (PBST) for 7 min. Slides were then treated with 3% H 2 O 2 for 5 min. After three 5-minute washes with PBST, the samples were blocked with PBS + 5% Donkey normal serum + 0.1% Triton X100 (PBSST) for 2 hrs at room temperature, and primary antibodies (see a complete list of primary antibodies used for this study in Table S3) diluted in PBSST were added. After being incubated overnight at 4°C, slides were washed five times with PBST (4 min per wash), then incubated with a secondary antibody (Invitrogen, 1:200) diluted in PBST for 2 hours at room temperature. The samples were washed three times in PBST (5 min per wash) and stained with DAPI or Hoechst 33342 (1μg/ml) diluted in PBST for 7 min. After five 4min washes with PBST and one 4-min wash with tap water, slides were mounted with mounting medium (VECTASHIELD) for imaging. Stained slides were stored at 4°C. All staining was performed on 3-8 hearts/group, with 2-3 sections/heart. To immunostain neonatal rat or mouse CMs following modRNA treatment, modRNA-transfected neonatal CMs were fixed on coverslips with 3.7% PFA for 15 min at room temperature, then washed three times with PBS. Following permeabilization with 0.5% Triton X in PBS for 10 min at room temperature, cells were blocked with 5% normal goat / donkey serum + 0.5% Tween 20 for 30 min. Coverslips were incubated with primary antibodies for 1 hr in a humid chamber at room temperature, followed by incubation with corresponding secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 647, and Alexa Fluor 555, as well as Hoechst 33342 staining for nuclei visualization (all from Invitrogen). The fluorescent images were taken on a Zeiss fluorescent microscope at 10X, 20X, and 40X magnification.

Western blot analysis
We isolated total protein from specific cells or tissues at given time points as mentioned above. In brief, equal amounts of protein were resolved using an SDS-PAG Electrophoresis system in 4%-15% Mini-PROTEAN TGX stain-free gels (Bio-Rad) and conjugate 1:3000, #12262; Cell Signaling) antibodies. Anti-rabbit and anti-mouse HRPconjugated secondary antibodies were purchased from Sigma-Aldrich. Antigen or antibody complexes were visualized with ChemiDoc Touch imaging system (Bio-Rad).

RNA isolation and gene expression profiling using real-time PCR
Total RNA was isolated using the RNeasy mini kit (Qiagen) and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time qPCR analyses were performed on a Mastercycler realplex 4 Sequence Detector (Eppendorf) using SYBR Green (Quantitect TM SYBR Green PCR Kit, Qiagen). Data were normalized to 18s expression where appropriate (endogenous controls). Fold changes in gene expression were determined by the ∂∂CT method and presented relative to internal control. PCR primer sequences are shown in Table S4.

Fetal, neonatal, or adult mouse CM isolation
CMs from the hearts of E18 fetal mice (Pip4k2c -/or wild type) or 3-to 4-day-old neonatal Sprague Dawley rats (Jackson) were isolated as previously described 62,63 .
We used multiple rounds of digestion with 0.14-mg/mL collagenase II (Invitrogen); after each, the supernatant was collected in horse serum (Invitrogen). The total cell suspension was centrifuged at 300 g for 5 min. Supernatants were discarded and cells were resuspended in DMEM (GIBCO) medium with 0.1mM ascorbic acid (Sigma), 0.5% Insulin-Transferrin-Selenium (100X), penicillin (100U/mL), and streptomycin (100μg/mL). Cells were plated in plastic culture dishes for 90 min until most of the non-CMs attached to the dish, and the CMs remained in suspension. The CMs were then seeded at 1 × 10 5 cells/well in a 24-well plate. Isolated CMs were incubated for 48 hrs in DMEM medium containing 5% horse serum plus Ara c. After incubation, cells were transfected with varying doses of different modRNAs, as described in the text. Adult CMs were isolated from mice using standard Langendorff's method as previously described 63 . To count CMs, we averaged three different samples and three hearts/group using a hemocytometer. We counted approximately 150-200 CMs/aliquot (10ul aliquot samples taken using a wide-bore pipette from the total volume of CMs obtained following digestion). The cultured CMs were stained with α-actinin (CM, Red) antibody (abcam) and Hoechst 33342 for nuclei counts. Approximately 1x10 3 CMs were used per sample for nuclei counts, using 3-4 independent samples per group.
Nuclei count was plotted as the percentage of counted CMs.

Assessing cardiomyocyte hypertrophy (CH)
To analyze CM hypertrophy, we used wheat germ agglutinin (WGA) staining. The cryofrozen heart sections were dried for 30-60 min at room temperature and dehydrated with PBS for 10 mins. Next, we applied the WGA (50µm) for 1 hr. The sections were washed three times with PBS for 5 mins each, and the mounting medium was applied.
To quantify CM size, images at 20X or 40X magnification were captured, and the ImageJ program was used to determine the area of each cell. Quantitative analyses involved counting multiple fields from three to six independent hearts/group and three sections/heart (~50 cells per field were assessed for a total ~250 cells per sample).

Sirius red / fast green staining
The Sirius red / fast green staining was performed according to standard procedures.
The cryo-frozen heart sections were dried for 1 hr at room temperature and incubated with Bouin's solution at 58°C for 1 hr, then washed with tap water for 5 mins to remove the yellow color from sections. The sections were stained with 0.1% fast green for 20 mins at room temperature and rinsed in 1% acetic acid for 1 min at room temperature.
Next, slides were washed with tap water for 5 mins. The sections were further stained with 0.1% Sirius red for 30 mins at RT. Slides were dehydrated sequentially by 70% ethanol for 30 sec, 100% ethanol for 1 min, and 100% xylene for 3 min, then covered with coverslips using Permount mounting medium (Fisher Chemical). The images were taken on a bright field microscope.

SB4311542 or rapamycin treatment in mice
For SB4311542 treatment, the stock solution of SB4311542 (10mM) was diluted into a vehicle in ethanol. Mice were given 10mg/kg/day of SB4311542 through i.v. injections every day for 23 consecutive days (from 2 days before TAC until the experiment ended (day 21)). For rapamycin treatment, the stock solution of rapamycin (50mg/mL) was diluted into a vehicle [5% (vol/vol) Tween-80, 5% (vol/vol) PEG 400 (polyethylene glycol, molecular weight 400)] in 1× PBS. Mice were given 3mg/kg/day of rapamycin through i.v. injections for 9 consecutive days (from 2 days before until 7 days post TAC).

Statistical analysis
Statistical significance was determined by unpaired two-tailed t-test, One-way ANOVA, Tukey's Multiple Comparison Test, Two-way ANOVA, Bonferroni post hoc test, or Logrank (Mantel-Cox) test for survival curves, as detailed in respective figure legends. p-Value<0.05 was considered significant. All graphs represent average values, and values were reported as mean ± standard error of the mean. An unpaired two-tailed ttest was based on assumed normal distributions. To quantify different parameters, we used WGA, pH3 CMs, and Sirius red / fast green staining with results acquired from at least three heart sections/heart in mouse numbers as noted in respective figure legends. Tables   Table S1:       body weight measurements (c) of E18 WT or KO-Pip4k2c mice (n=5). d.

Supplemental
Representative images of the whole heart. e&f. Heart weight to body weight ratio (e, n=5) and quantification of isolated CM (f) from the hearts of E18 WT or KO-Pip4k2c mice (n=3). Unpaired two-tailed t-test. N.S., Not Significant. Scale bar = 1mm (d).