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

Figure 1. PIP4K2C and Pip4k2c expression decreases in failing hearts of humans and mice, respectively. a) PIP4K2C mRNA expression in human non‐injured normal left ventricle (LV) (non‐failing, NF, n = 5), cardiac hypertrophy (CH, n = 5), and heart failure (HF, n = 5). b. Western blot of PIP4K2C expression in human LV samples with GAPDH as control (n = 4 for NF, n = 5 for CH, n = 5 for HF). c) Quantitative analysis of b. d) Experimental timeline to analyze Pip4k2c expression in sham‐operated or TAC mouse model. e) mRNA expression of Pip4k2c in WT mouse heart samples compared to those from TAC recipients, 4 days after injury (n = 4). f) Western blot evaluation of Pip4k2c protein in WT mouse heart samples compared to those from TAC recipients 4 days after injury, with GAPDH as a control housekeeping gene. g) Quantitative analysis of Pip4k2c western blot from f (n = 2). h) Pip4k2c mRNA expression in CM or non‐CMs after Sham or 7 or 21 days post TAC injury (n = 3). One‐way ANOVA, Tukey's Multiple Comparison Test were used in a,c; unpaired two‐tailed t‐test was used in e,g; and two‐way ANOVA and Bonferroni post‐hoc tests were used in h. ****, P < 0.0001, ***, P < 0.001, **, P < 0.01.

Figure 2. Loss of Pip4k2c enhances formation of cardiac hypertrophy and fibrosis post TAC injury. a) Experimental timeline to evaluate cardiac function and outcome in Pip4k2c−/− (KO‐Pip4k2c) and Pip4k2c+/+ littermate controls (WT) in a TAC mouse model. b) Representative echocardiography image of left ventricle 21 days post sham or TAC injury in WT or KO‐Pip4k2c. c–f) Echo evaluation of delta % left ventricular ejection fraction (c), fractioning shorting (d), LVIDd (e), and LVIDd (f) 21 days post sham or TAC injury in WT or KO‐Pip4k2c (n = 10). g) Heart weight to tibia length 21 days post sham or TAC injury in WT or KO‐Pip4k2c (n = 10). h,i) Representative images of whole heart (h) and H&E staining (i) 21 days post sham or TAC injury in WT or KO‐Pip4k2c. j) Representative images of wheat germ agglutinin (WGA) staining to evaluate CM size (cross‐sectional area) 21 days post sham or TAC injury in WT or KO‐Pip4k2c. k) Quantitative analysis of j (n = 8). l,m) qPCR analysis of hypertrophic markers 21 days post sham or TAC injury in WT or KO‐Pip4k2c (n = 5). n) Representative images of Sirius red / fast green indicating fibrotic area 21 days post sham or TAC injury in WT or KO‐Pip4k2c. o)…

Figure 3. Pip4k2c modRNA elevates Pip4k2c levels, attenuating cardiac hypertrophy and fibrosis post TAC injury. a) Experimental timeline to evaluate Pip4k2c modRNA expression and cardiac function in TAC mouse model. b) Western blot of Luc (control) or Pip4k2c modRNA expression in mouse hearts 24 hours post TAC (n = 2) and quantitative analysis thereof (n = 2). c. Representative echocardiography image of left ventricle 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA. d–g) Echo evaluation of delta % left ventricular ejection fraction (d), fractioning shorting (e), LVIDd (f), and LVIDs (g) before (day 0), 14, or 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA (n = 8). h) Representative images of whole heart 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA. i) Heart weight to tibia length 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA (n = 8). j) Representative images of WGA staining to evaluate CM size (cross‐sectional area) 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA. k) Quantitative analysis of j (n = 8). l) qPCR analysis of hypertrophic markers 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA (n = 5). m)…

Figure 4. Pip4k2c attenuates cardiac hypertrophy and fibrosis via mTORC1 and TGFβ1, respectively. a) Experimental timeline to evaluate mTORC1 pathway activation. b) Western blot analysis of phospho‐p70s6k and p70s6k protein expression in WT or KO‐Pip4k2c 21 days post sham or TAC injury. c) Quantitative analysis of b (n = 2). d) Experimental timeline to evaluate the effect of vehicle or rapamycin small molecule on cardiac function, CH, and CF. e) Representative echocardiography image of left ventricle 21 days post TAC injury and delivery of vehicle or rapamycin. f–h) Echo evaluation of fractioning shorting (f), LVIDd (g), and LVIDd (h) 21 days post TAC injury and delivery of vehicle or rapamycin (n = 4). i) Representative images of whole heart 21 days post TAC injury and delivery of vehicle or rapamycin. j,k) Heart weight (j) or lung weight (k) to tibia length 21 days post TAC injury and delivery of vehicle or rapamycin (n = 4). l) Representative images of WGA staining to evaluate CM size (cross‐sectional area) 21 days post TAC injury and delivery of vehicle or rapamycin. m. Quantitative analysis of l (n = 4). n) qPCR analysis of ANP and BNP 21 days post TAC injury and delivery of vehicle…

Figure 5. Pip4k2c attenuates cardiac fibrosis via the TGFβ1 pathway. a) Experimental timeline to evaluate TGFβ pathway activation. b) Western blot analysis of TGFβ protein expression in WT or KO‐Pip4k2c 21 days post sham or TAC injury. c) Quantitative analysis of b (n = 2). d) Experimental timeline to evaluate the effect of vehicle or SB4311542 small molecule on cardiac function and CF. e) Representative echocardiography image of left ventricle 21 days post TAC injury and delivery of vehicle or SB4311542. f–h) Echo evaluation of fractioning shorting (f), LVIDd (g), or LVIDd (h) 21 days post TAC injury and delivery of vehicle or SB4311542 (n = 3). i) Heart weight to tibia length 21 days post TAC injury and delivery of vehicle or SB4311542 (n = 3). j) Lung weight to tibia length 21 days post TAC injury and delivery of vehicle or SB4311542 (n = 3). k) Representative images of Sirius red / fast green to evaluate fibrotic area 21 days post TAC injury and delivery of vehicle or SB4311542. l) Quantitative analysis of k (n = 3). m) A proposed model for regulating Pip4k2c modRNA in CH and CF by inhibiting mTORC1 and TGFβ1 post TAC injury. One‐way ANOVA, Tukey's Multiple Comparison Test were used in…

Figure 6. Pip4k2c suppresses the TGFβ1 pathway via its N‐terminal motif (VMLLPDD), which inhibits Pip5k1α, phospho‐AKT 1/2/3, and phospho‐Smad3. a) Experimental plan to evaluate the impact of mutant Pip4k2c (original N‐terminal motif VMLLPDD replaced with EIFLPNN) on cardiac function in the TAC mouse model. b) modRNA strategy for the three modRNA tested. c–f) Echo evaluation of delta % left ventricular ejection fraction (c), fractioning shorting (d), LVIDd (e), and LVIDd (f) 21 days post TAC injury and delivery of Luc, Pip4k2c, or mutant Pip4k2c modRNA (Luc‐, n = 3; Pip4k2c‐ n = 3; mutant Pip4k2c modRNA‐ n = 6). g) Heart weight to tibia length 21 days post TAC injury and delivery of Luc or Pip4k2c modRNA (Luc‐, n = 3; Pip4k2c‐ n = 3; mutant Pip4k2c modRNA‐ n = 6). h) Experimental plan to evaluate Pip4k2c's effect on fibrosis marker expression in cardiac fibroblasts isolated and sorted (for the fibroblastic marker CD90) from WT or KO‐Pip4k2c hearts 21 days post TAC injury and treated with DMSO (control), TbetaR1/ALK5 inhibitor (SB431542), Pip4k2c, or mutant Pip4k2c modRNA i) qPCR analysis of fibrosis markers placed downstream of TGFβ1 following different treatments (n = 3). j) Estimation of…