Graphical abstract graphic file with name fx1.jpg [59]Open in a new tab Highlights * • Liver-heart crosstalk orchestrates pressure-driven left ventricular hypertrophy * • Hepatic FGF21 increase followed by cardiomyocyte FGF21 induction drives hypertrophy * • Oxytocin attenuates the pro-hypertrophic effect of FGF21 * • Systemic or cardiomyocyte-specific inhibition of FGF21 holds therapeutic value __________________________________________________________________ FGF21, a mainly liver-derived hormone, has multiple beneficial metabolic effects. Sobuj Mia et al. reveal a pathologic FGF21-based hepato-cardiac crosstalk mechanism that is induced with pressure overload and leads to left ventricular hypertrophy. Hepatocyte-derived FGF21 triggers cardiomyocyte FGF21 expression, which accounts for hypertrophy. Thus, FGF21 inhibition holds therapeutic potential. Introduction Heart failure (HF) still constitutes a major cause of mortality and hospital admissions despite decades of efforts for the improvement of applied treatments. In many cases, HF is accompanied by left ventricular (LV) hypertrophy (LVH). LVH is an early adaptive mechanism in response to increased heart workload, which can progress to detrimental cardiac complications associated with high cardiovascular morbidity and mortality.[60]^1 The increase in myocardial wall thickness allows the heart to counterbalance pressure-induced wall stress that may be caused from physiological stressors, such as exercise or from pathological events such as aortic stenosis or uncontrolled hypertension,[61]^2 and eventually causes systolic and diastolic myocardial dysfunction.[62]^3^,[63]^4 While various intracellular signaling pathways that are involved in cardiomyocyte (CM) enlargement and LVH are known, the systemic mechanisms that ignite the onset of the disease remain partially characterized. Fibroblast growth factor (FGF) 21 is an endocrine hormone that regulates systemic energy homeostasis. It is mainly produced in the liver,[64]^5^,[65]^6 but skeletal muscle,[66]^7 white adipose tissue (WAT),[67]^8 brown adipose tissue,[68]^9 and the pancreas[69]^10 also contribute to the systemic FGF21 levels. Various studies, including one of ours,[70]^11 suggest that the heart can also serve as a source of FGF21.[71]^11^,[72]^12 FGF21[73]^5^,[74]^6^,[75]^13^,[76]^14^,[77]^15 signals through the β-klotho/FGF receptor (FGFR)1c complex.[78]^16 Pharmacological administration of FGF21 in animal models of diabetes and obesity, as well as treatment of obese humans with FGF21 analogs,[79]^17^,[80]^18 normalized circulating glucose and lipids and lowered body weight[81]^17^,[82]^18^,[83]^19^,[84]^20 and hepatic fat.[85]^21 While the metabolic benefits of FGF21 have been translated to clinical applications, the effects of prolonged FGF21 signaling to the heart remain controversial. Stimulation of cardiac FGF21 production has been described as an adaptive response to physiological stress.[86]^12 On the other hand, increased FGF21 signaling has been associated with cardiac dysfunction in patients with end-stage HF,[87]^22 as well as with diastolic dysfunction and higher mortality in patients with HF with preserved ejection fraction (HFpEF),[88]^23 hypertension,[89]^24 dilated cardiomyopathy,[90]^25 coronary artery disease,[91]^26^,[92]^27^,[93]^28 acute myocardial infarction,[94]^28 atrial fibrillation,[95]^29 and type 2 diabetes.[96]^30 The dual effects of FGF21 on metabolism and heart health led us to explore its role in stress-induced HF. Our study identified a hepato-cardiac signaling pathway that accounts for LVH and HF that are induced with pressure overload (PO). This pathway involves FGF21 production in the liver, which targets the heart, leading to CM FGF21 production that drives hypertrophy via a previously unknown mechanism that seems to interfere with the cardioprotective signaling of hypothalamus-derived oxytocin. Results Blood and cardiac FGF21 levels are increased in human patients and mice with cardiac hypertrophy or increased afterload Recently, it was shown that circulating levels of FGF21 are substantially elevated in patients with end-stage HF, as well as that this change coincided with higher FGF21 deposition in myocardium samples.[97]^22 To explore further whether FGF21 is involved in HF, we assessed serum FGF21 levels in humans with LVH ([98]Table S1) or hypertension ([99]Table S2). Neither of the patient groups has developed systolic dysfunction, which occurs with cardiac hypertrophy. Our analyses identified higher serum FGF21 levels in both patient groups compared to control groups without LVH ([100]Figure 1A) or those without hypertension ([101]Figure 1B), respectively. To investigate if observations that correlated high serum FGF21 levels with elevated myocardial FGF21 abundance in patients with end-stage HF[102]^22 apply in patients with increased afterload, we measured FGF21 expression in heart tissue samples from both male and female individuals with systemic hypertension ([103]Table S3) (samples were obtained from the National Disease Research Interchange). Our analysis showed significantly higher FGF21 mRNA levels (15-fold) in hearts of hypertensive individuals compared to normotensives ([104]Figure 1C). Thus, humans with increased afterload or LVH have higher serum and cardiac FGF21 content even prior to the development of systolic dysfunction. Figure 1. [105]Figure 1 [106]Open in a new tab Hepatic and cardiac FGF21 expression is increased in humans and mice with increased afterload Cardiac hypertrophy, cardiac function, and fibrosis-related gene expression changes during progression of cardiac hypertrophy. (A–C) Serum FGF21 (A and B) and cardiac FGF21 mRNA levels (C) in humans with left ventricular hypertrophy (A) or hypertension (B and C), compared to individuals without hypertrophy or normotensive individuals. (D–F) Heart weight normalized to tibia length (D), representative short-axis M-Mode images of the left ventricle (E), and measurements of fractional shortening (F), in C57BL/6 mice subjected to control sham surgery or TAC for 3 days (no cardiac function data), 2 weeks, 4 weeks, and 8 weeks post surgery. (G–K) Cardiac COL1A1, COL3A1, PSTN, β-MHC, and BNP mRNA levels in the C57BL/6 mice subjected to sham or TAC for 3 days, 2 weeks, 4 weeks, and 8 weeks (G), plasma FGF21 (H), FGF21 mRNA in the liver (I), heart (J), and white adipose tissue (K) in C57BL/6 mice subjected to control sham surgery or TAC for 3 days (no cardiac function data), 2 weeks, 4 weeks, and 8 weeks post surgery. (A) Healthy (no LVH), n = 45; hypertrophy, n = 31. (B) Normotensive, n = 18; hypertensive, n = 50. (C) Normotensive, n = 6; hypertensive, n = 6. (D) Sham, n = 8; 3 days TAC, n = 12; 2 weeks TAC, n = 9; 4 weeks TAC, n = 10; 8 weeks TAC, n = 11. (E) Sham, n = 7; 2 weeks TAC, n = 7; 4 weeks TAC, n = 7; 8 weeks TAC, n = 7. (F) Sham, n = 7; 2 weeks TAC, n = 7; 4 weeks TAC, n = 7; 8 weeks TAC, n = 7. (G) Sham, n = 5–6; 3 days TAC, n = 4–8; 2 weeks TAC, n = 6–8; 4 weeks TAC, n = 6–8; 8 weeks TAC, n = 6–8. (H) Sham, n = 11; 3 days TAC, n = 10; 2 weeks TAC, n = 10; 4 weeks TAC, n = 9; 8 weeks TAC, n = 12. (I) Sham, n = 8; 3 days TAC, n = 9; 2 weeks TAC, n = 11; 4 weeks TAC, n = 9; 8 weeks TAC, n = 9. (J) Sham, n = 9; 3 days TAC, n = 12; 2 weeks TAC, n = 12; 4 weeks TAC, n = 9; 8 weeks TAC, n = 10. (K) Sham, n = 8; 3 days TAC, n = 6; 2 weeks TAC, n = 7; 4 weeks TAC, n = 7; 8 weeks TAC, n = 6. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using one-way or two-way ANOVA with Tukey multiple comparison test or Student’s t test when applicable. These observations motivated us to test whether elevation in serum FGF21 constitutes an adaptive or maladaptive response to increased cardiac afterload. To this end, we investigated how FGF21 production changes over the course of the development of LVH in a mouse model of increased LV afterload (transverse aortic constriction [TAC]). We applied TAC for 3 days, 2 weeks, 4 weeks, and 8 weeks ([107]Figure S1A for TAC-induced pressure gradient data) in wild-type mice, which induced cardiac hypertrophy ([108]Figure 1D) and cardiac dysfunction ([109]Figures 1E and 1F; [110]Table S4) and increased cardiac expression of the fibrosis markers, collagen type I alpha 1 (COL1A1), COL3A1, periostin (PSTN), and the HF markers β-myosin heavy chain (β-MHC) and B-type natriuretic peptide (BNP) ([111]Figure 1G). PO increased plasma FGF21 levels ([112]Figure 1H) in both male and female mice ([113]Figure S1B) that coincided with higher expression of FGF21 in the liver ([114]Figure 1I) in both males and females ([115]Figure S1C). In accordance with our findings in humans ([116]Figure 1C), both male and female mice with PO had higher cardiac FGF21 expression ([117]Figures 1J and [118]S1D). On the other hand, FGF21 expression did not increase either in the skeletal muscle ([119]Figure S1E) or in the brown adipose tissue ([120]Figure S1F), although it was increased in WAT in both male and female mice ([121]Figures 1K and [122]S1G). Thus, PO is associated with higher systemic FGF21 levels and increased expression in the liver, WAT, and the heart. Figure 2. [123]Figure 2 [124]Open in a new tab Changes in cardiac transcriptome, metabolome, and lipidome occur as soon as 3 days post TAC and continue throughout the progression of cardiac hypertrophy Volcano plot (A–C), hierarchical clustering (D–F), KEGG pathway analysis (G), and summary of metabolomic and lipidomic changes (H) depicting upregulation (A–H) or downregulation (A–H) in gene expression (A–G), lipids, and metabolites (H) in hearts obtained from C57BL/6 mice subjected to control sham surgery or TAC 3 days (A, D, and H), 2 weeks (B, E, and H), and 8 weeks (C, F, G, and H) post surgery. Sham, n = 5; TAC, n = 5. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using Student’s t test. To explore whether upregulation of hepatic and cardiac FGF21 expression constitutes a protective adaptive response to PO or a cause for the development of cardiac hypertrophy and systolic dysfunction, we characterized the temporal profile of FGF21 expression in relation to the onset of cardiac dysfunction. Hepatic FGF21 expression ([125]Figure 1I) and plasma FGF21 levels ([126]Figure 1H) increased as early as 3 days post TAC (8.3-fold for plasma FGF21) and remained elevated up to 8 weeks (4.2-fold for plasma FGF21) in both males and females ([127]Figures S1B and [128]1C). Although hepatic and plasma FGF21 upregulation constitutes an early response to PO, the increase in cardiac FGF21 expression occurred with a delay (2 weeks post TAC) ([129]Figures 1J and [130]S1D), coinciding with cardiac hypertrophy ([131]Figure 1D), dysfunction ([132]Figures 1E and 1F; [133]Table S4), and upregulation of cardiac fibrosis-related and HF-related (BNP and β-MHC) gene expression ([134]Figure 1G), which has been associated with pathological cardiac hypertrophy.[135]^31 Interestingly, the increase of FGF21 expression in WAT was observed even later (4 weeks post TAC), after cardiac dysfunction and hypertrophy had occurred ([136]Figure 1K). Conclusively, PO instantly stimulates FGF21 expression in the liver and release of FGF21 in the circulation, which are followed later by induction of cardiac FGF21 that coincides with the onset of LVH and left ventricular dysfunction and upregulation of WAT FGF21 that follows cardiac complications. FGF21 elevation is accompanied by myocardial fibrosis, metabolic derangement, and accumulation of toxic lipid species To assess the gene expression changes that FGF21 stimulates in the heart over the course of cardiac hypertrophy progression, transcriptomic analysis was conducted in mouse hearts at 3 days, 2 weeks, and 8 weeks post TAC. A total of 1,831 genes, 1,717 genes, and 1,186 genes were differentially expressed at 3 days, 2 weeks, and 8 weeks post TAC, respectively. Volcano plot ([137]Figures 2A–2C) and hierarchical clustering ([138]Figures 2D–2F) analyses identified distinct transcriptomic profiles among the 3 experimental groups. Furthermore, genes that are involved in fibrosis and hypertrophy were among the top 50 differentially expressed genes in the early (Col3a1, Col8a1, Fbn, Col15a1, Nppa, and Dpysl3) and late (Acta1, Ankrd1, and Flnc) phases of hypertrophy progression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed consistent upregulation of cardiac extracellular matrix (ECM)-interaction pathways across all time points ([139]Figures 2G and [140]S2A–S2C). Notably, 2 weeks and 8 weeks post TAC, there was a pronounced increase in gene expression of components of pathways associated with phosphatidylinositol 3-kinase (PI3)-AKT, dilated cardiomyopathy, and hypertrophic cardiomyopathy signaling ([141]Figures S2B and S2C). In contrast, genes involved in branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine or catabolism of fatty acid were downregulated across all time points ([142]Figures S2D–S2F). To corroborate our transcriptional profiling with the metabolic profile of the heart, we performed metabolomic and lipidomic analyses in the same sets of samples at 3 days, 2 weeks, and 8 weeks post-TAC. Metabolomic analysis showed that 3 days post TAC, myocardial glucose content is significantly lower compared to control mice with sham surgery and so is the glucose derivative, pyruvate, as well as citrate and NAD^+ ([143]Figure 2H; [144]Table S5A). On the other hand, β-hydroxybutyrate (ketone body) seems to accumulate 3 days post TAC ([145]Figure 2H; [146]Table S5A). At 2 weeks post TAC, glucose levels remain lower compared to sham ([147]Figure 2H; [148]Table S5B). Conversely, succinate, as well as derivatives of glutamine and glutamate metabolism, such as asparagine and serine, are increased ([149]Figure 2H; [150]Table S5B). Notably, 8 weeks post TAC, when pathological hypertrophy has fully developed, cardiac BCAAs (valine, leucine, and isoleucine) accumulate, as do serine, asparagine, and glutathione (another derivative of glutamate) ([151]Figure 2H; [152]Table S5C). Accumulation of these nutrients is accompanied by lower levels of both NAD^+ and ATP, implying lower electron transport toward oxidative phosphorylation and ATP synthesis. Lipidomic analysis showed that although total lipid species do not change significantly, certain lipid species are altered at various time points ([153]Figure 2H; [154]Table S6). At 3 days post TAC, triglycerides (TGs) increased while diacylglycerols (DAGs) decreased, suggesting reduced TG hydrolysis ([155]Figure 2H; [156]Table S6A). At 2 weeks post TAC, 14 TG and 2 DAG species decreased, while phospholipid species (phosphatidylcholine [PC], phosphatidylethanolamine [PE], and phosphatidylserine [PS]) increased ([157]Figure 2H; [158]Table S6B), likely indicating a higher demand for membrane synthesis as hypertrophy begins ([159]Figure 1D). At 8 weeks post TAC, phospholipid species (13 PC, 12 PE, and 2 PS) remain elevated ([160]Figure 2H; [161]Table S6C). Cardiac lipidome changes at this late stage include acetyl-carnitine accumulation (precursor of carnitine; carnitine facilitates transportation of fatty acyl-CoAs in mitochondria for β-oxidation), reduced fatty acyl-carnitines, and decreased cardiolipin levels, a phospholipid of the inner mitochondrial membrane ([162]Figure 2H; [163]Table S6C). Overall, combined transcriptomic, metabolomic, and lipidomic analysis shows that cardiac hypertrophy progression involves reprogramming of metabolic pathways including early β-hydroxybutyrate accumulation and glucose depletion, followed by increased BCAAs, phospholipids, and glutamine pathway byproducts, alongside reduced acyl-carnitines, NAD^+, and ATP at later stages. Hepatic FGF21 induction is associated with liver congestion To determine whether hepatic congestion from TAC induces FGF21 expression, we used partial inferior vena cava ligation (pIVCL) as an alternative model. Mice with pIVCL showed significant upregulation of hepatic FGF21 mRNA levels compared to control sham mice ([164]Figure S2G). Further analyses in mice with either TAC or pIVCL showed a significant increase of the hepatic gene expression of the mechano-sensors leucine-rich repeat-containing protein (LRRC)8A, LRRC8B, and Piezo-type mechanosensitive ion channel component (PIEZO)1 ([165]Figures S2H–S2M). Thus, hemodynamic congestion in the liver increases FGF21 expression, which is associated with elevated expression of mechano-sensor proteins. Inhibition of hepatic FGF21 lowers circulating FGF21 levels and alleviates cardiac hypertrophy with PO To determine whether FGF21 upregulation is a cause or consequence of HF in TAC mice, we studied hepatocyte-specific FGF21 knockout (HEP-FGF21^−/−) mice that we generated by crossing Alb-Cre^+/− with Fgf21^fl/fl mice. Since hepatic FGF21 upregulation precedes cardiac hypertrophy and LV dysfunction, we examined the effects of PO in these mice. First, we confirmed that deletion of FGF21 in the liver lowered hepatic FGF21 expression ([166]Figure S3A) without affecting cardiac or WAT FGF21 levels ([167]Figures S3B and S3C). Control groups (Fg21^fl/fl, Alb-Cre^+/−, and αMHC-Cre^+/− mice) showed similar responses to TAC surgery as shown by heart weight/tibia length (HW/TL) ([168]Figure S3D), systolic dysfunction ([169]Figure S3E), and expression of β-MHC ([170]Figure S3F) and COL3A1 ([171]Figure S3G), allowing us to combine floxed and Cre-expressing mice as controls. Then, we applied TAC in HEP-FGF21^−/− and control mice for 8 weeks and confirmed equivalent increase in pressure gradient ([172]Figure S4A). Hepatocyte-specific deletion of FGF21 prevented elevation of FGF21 expression in the liver ([173]Figure 3A) and maintained plasma FGF21 levels within the normal range ([174]Figure 3B) when TAC was applied. Strikingly, HEP-FGF21^−/− mice showed less profound cardiac hypertrophy ([175]Figure 3C), and cardiac function was protected ([176]Figures 3D and 3E; [177]Table S7). Notably, no male or female-specific differences were observed in the HEP-FGF21^−/− mice following TAC, either in circulating FGF21 or tissue expression of FGF21 or in cardiac function and hypertrophic response as shown by hepatic FGF21 expression ([178]Figure S4B), circulating FGF21 levels ([179]Figure S4C), HW/TL ([180]Figure S4D), and cardiac function ([181]Figure S4E). Alleviation of cardiac hypertrophy and improvement of heart function in HEP-FGF21^−/− mice were accompanied by lower cardiac expression of HF markers BNP ([182]Figure 3F) and β-MHC ([183]Figure 3G), attenuation of CM enlargement ([184]Figures 3H and [185]S4F), less myocardial fibrosis ([186]Figures 3H and [187]S4G), reduced fibrosis-related gene expression (COL1A1, COL3A1, and PSTN) ([188]Figures S5A–S5C), lower cardiac expression ([189]Figures S5D–S5F), and circulating levels ([190]Figures S5G–S5I) of the inflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor alpha (TNF-α), while IL-10 was not altered ([191]Figures S5J and S5K) compared to control mice with TAC. Accordingly, pro-hypertrophic signaling was attenuated in hearts of HEP-FGF21^−/− mice with TAC, as shown by lower pAMPK/tAMPK ([192]Figures 3I and [193]S5L) and pERK/tERK ratio ([194]Figures 3I and [195]S5M) compared to control mice with TAC. Figure 3. [196]Figure 3 [197]Open in a new tab Genetic ablation of hepatocyte FGF21 lowers cardiac FGF21 expression and attenuates cardiac hypertrophy and dysfunction (A–J) Hepatic FGF21 mRNA (A), plasma FGF21 (B), heart weight normalized to tibia length (C); representative short-axis M-mode images of the left ventricle (D); measurements of fractional shortening (E); cardiac BNP (F) and β-MHC (G) mRNA levels; WGA staining (H, upper; scale bar, 100 μm); PSR staining (H, lower; scale bar, 100 μm); cardiac FGF21, pAMPK^Thr172, AMPK, pERK1/2, ERK, PPARα, SIRT1, pAKT^Ser473, and β-actin protein levels (I); and cardiac FGF21 mRNA (J) in control (floxed or Alb-Cre^+/−) and HEP-FGF21^−/− mice subjected to control sham surgery or TAC 8 weeks post surgery. (K–M) Phalloidin and DAPI staining (scale bar, 100 μm) (K), quantification of CM cell size (L), and FGF21 mRNA (M) of adult primary CM treated either with rm-FGF21 or control vehicle for 24 h. (N–P) Cardiac FGFR1 mRNA levels in C57BL/6 mice subjected to sham surgery or TAC for 3 days, 2 weeks, 4 weeks, and 8 weeks (N), in humans with hypertension or normotensive individuals (O) and in control or HEP-FGF21^−/− mice subjected to sham or TAC surgery 8 weeks post surgery (P). (A) Ctrl sham, n = 6; ctrl TAC, n = 6; HEP-FGF21^−/− TAC, n = 6. (B) Ctrl sham, n = 6; ctrl TAC, n = 7; HEP-FGF21^−/− TAC, n = 7. (C) Ctrl sham, n = 9; ctrl TAC, n = 10; HEP-FGF21^−/− TAC, n = 9. (D and E) Ctrl sham, n = 6; ctrl TAC, n = 6; HEP-FGF21^−/− TAC, n = 6. (F) Ctrl sham, n = 6; ctrl TAC, n = 6; HEP-FGF21^−/− TAC, n = 6. (G) Ctrl sham, n = 6; ctrl TAC, n = 7; HEP-FGF21^−/− TAC, n = 6. (H) Ctrl sham, n = 6; ctrl TAC, n = 6; HEP-FGF21^−/− TAC, n = 6. (I) Ctrl sham, n = 3–6; ctrl TAC, n = 5–11; HEP-FGF21^−/− TAC, n = 6–10. (J) Ctrl sham, n = 7; ctrl TAC, n = 7; HEP-FGF21^−/− TAC, n = 7. (K and L) Vehicle, n = 5; rmFGF21, n = 6. (M) Vehicle, n = 6; rmFGF21, n = 6. (O) Normotensive, n = 5; hypertensive, n = 6. (P) Ctrl sham, n = 7; ctrl TAC, n = 9; HEP-FGF21^−/− TAC, n = 10. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using one-way ANOVA with Tukey multiple comparison test or Student’s t test when applicable. Surprisingly, the hearts of HEP-FGF21^−/− mice showed almost complete prevention of FGF21 mRNA ([198]Figure 3J) and protein ([199]Figures 3I and [200]S5N) induction with TAC. On the other hand, no significant changes were observed in FGF21 mRNA levels in the skeletal muscle ([201]Figure S5O) and brown adipose tissue ([202]Figure S5P). However, the TAC-induced FGF21 upregulation in WAT seen in controls ([203]Figure 1K) was absent in HEP-FGF21^−/− mice ([204]Figure S5Q). Liver-derived FGF21 controls CM FGF21 expression The similar pattern of cardiac and hepatic FGF21 expression prompted us to determine whether this was due to improved cardiac function or direct effects of circulating FGF21 on CMs. To this end, we treated primary CMs, isolated from adult mice, and a human CM cell line (AC16) with recombinant mouse (rm) and recombinant human (rh) FGF21, respectively, in vitro. In both cases, FGF21 promoted CM enlargement ([205]Figures 3K, 3L, [206]S6A, and S6B) and stimulated endogenous FGF21 expression ([207]Figures 3M and [208]S6C–S6E). The pro-hypertrophic effect of FGF21 was accompanied by increased pERK/tERK ratio ([209]Figures S6F and S6G). Thus, inhibition of hepatic FGF21 alleviates PO-induced hypertrophy, which is associated with suppression of cardiac FGF21 expression that seems to be driven by the lower circulating FGF21 levels. To explore further how CMs sense exogenous FGF21 signaling, we assessed the expression of the main CM FGF21 receptors (FGFR). This analysis showed that cardiac FGFR1 expression was significantly upregulated 2 weeks post TAC when cardiac FGF21 was also increased ([210]Figure 3N), and it was further elevated 4 and 8 weeks post TAC ([211]Figure 3N). Conversely, the expression of another important cardiac FGF21 receptor, FGFR4, was gradually suppressed as LVH progressed ([212]Figure S6H) and so were β-klotho mRNA levels ([213]Figure S6I). Accordingly, FGFR1 expression was elevated, while FGFR4 expression was reduced in myocardial tissue from individuals with hypertension [214](Figures 3O and [215]S6J) and in AC16 cells treated with rhFGF21 ([216]Figures S6K and S6L). Notably, cardiac FGFR1 expression was not upregulated in HEP-FGF21^−/− mice with TAC ([217]Figure 3P). Thus, FGF21-mediated LVH is associated with increased FGFR1 expression. As FGF21 expression is regulated by PPARα,[218]^5^,[219]^6 Sirtuin (SIRT),[220]^12^,[221]^32 and Akt signaling,[222]^7 we measured their cardiac protein levels in the control mice with TAC, as well as in HEP-FGF21^−/− mice. These analyses showed that cardiac PPARα ([223]Figures 3I and [224]S6M), SIRT1 ([225]Figures 3I and [226]S6N), and pAkt/tAkt ([227]Figures 3I and [228]S6O) expression was increased in control mice with TAC, which was prevented ([229]Figures 3I and [230]S6M–S6O) in HEP-FGF21^−/− mice with lower plasma FGF21 levels and protection from TAC-induced LVH. Thus, systemic FGF21 upregulation that follows PO activated cardiac PPARα, SIRT1, and pAkt/tAkt signaling, which was reversed by normalizing plasma FGF21 levels. CM FGF21 is the primary driving force of PO-induced hypertrophy and HF To investigate whether CM FGF21 upregulation per se is involved in the pathophysiology of cardiac hypertrophy with PO, we generated CM-specific FGF21 knockout mice (CM-FGF21^−/−) by crossing αMHC-Cre^+/− mice with Fgf21^fl/fl mice and confirmed FGF21 deletion in CMs without affecting hepatic or WAT FGF21 ([231]Figures S7A–S7C). Application of TAC in CM-FGF21^−/− mice for 8 weeks ([232]Figure S7D) did not increase cardiac FGF21 expression although there was a statistically non-significant trend of increase in both male and female mice ([233]Figure 4A; [234]Figure S7E), despite the elevation of hepatic FGF21 expression ([235]Figure S7F) and plasma FGF21 levels ([236]Figure 4B). Importantly, CM FGF21 ablation prevented the onset of cardiac hypertrophy ([237]Figure 4C) and cardiac dysfunction ([238]Figures 4D and 4E; [239]Table S7). CM-FGF21^−/− mice with TAC did not present any male or female-specific differences either in hepatic ([240]Figure S7F) or circulating ([241]Figure S7G) levels of FGF21 or in hypertrophy ([242]Figure S7H) and cardiac function ([243]Figure S7I). Accordingly, CM-FGF21^−/− mice with TAC had lower expression of BNP ([244]Figure 4F) and β-MHC ([245]Figure 4G), less CM enlargement ([246]Figures 4H and 4I) and myocardial fibrosis ([247]Figures 4H and 4J), and reduced fibrosis-related gene expression of COL1A1, COL3A1, and PSTN ([248]Figures S7J–S7L). Cardiac expression ([249]Figures S8A–S8C) and plasma content ([250]Figures S8D–S8F) of inflammatory cytokines, IL-1α, IL-β, and TNF-α, were markedly decreased in CM-FGF21^−/− mice compared to Fgf21^fl/fl mice with TAC. The anti-inflammatory IL-10 cardiac mRNA and plasma levels remained unchanged ([251]Figures S8G and S8H). Furthermore, besides lower cardiac FGF21 protein content ([252]Figures 4K and [253]S8I), CM-FGF21^−/− mice with TAC had lower pAMPK/tAMPK ([254]Figures 4K and [255]S8J) and pERK/tERK ([256]Figures 4K and [257]S8K), implying inactivation of pro-hypertrophic signaling pathways, compared to control mice with TAC. Notably, cardiac protein abundance of the FGF21 expression regulators, PPARα ([258]Figures 4K and [259]S8L) and pAkt/tAkt ([260]Figures 4K and [261]S8M), was either partially suppressed or remained elevated in CM-FGF21^−/− mice. On the other hand, cardiac SIRT1 expression was restored to normal levels in CM-FGF21^−/− mice ([262]Figures 4K and [263]S8N). Moreover, cardiac FGFR1 expression was not upregulated in CM-FGF21^−/− mice with TAC ([264]Figure 3L) as we observed in HEP-FGF21^−/− mice ([265]Figure 3P). However, the expression of β-KLOTHO mRNA was suppressed by ∼50% in both control and CM-FGF21^−/− mice with TAC ([266]Figure S8O). Conclusively, despite increased of hepatocyte-derived FGF21, CM-specific ablation of Fgf21 recapitulates the cardioprotective effects observed in HEP-FGF21^−/−, mice although signaling that promotes FGF21 expression is not completely blocked. Thus, stimulation of CM FGF21 expression with PO is a critical molecular event that accounts for cardiac hypertrophy and HF. Figure 4. [267]Figure 4 [268]Open in a new tab Genetic deletion of CM FGF21 prevents cardiac hypertrophy independent of systemic FGF21 levels Cardiac FGF21 mRNA (A), plasma FGF21 (B), heart weight normalized to tibia length (C); representative short-axis M-mode images of the left ventricle (D); measurements of fractional shortening (E); cardiac BNP (F) and β-MHC mRNA levels (G); WGA staining (H, upper; scale bar, 100 μm); quantification of cell size area (I); PSR staining (H, lower; scale bar, 100 μm); quantification of the fibrotic tissue area (J); cardiac FGF21, pAMPK^Thr172, AMPK, pERK1/2, ERK, PPARα, pAKT^Ser473, AKT, SIRT1, and β-actin protein (K); and cardiac FGFR1 mRNA in control (combined floxed and αMHC-Cre^+/−) and CM-FGF21^−/− mice subjected to control sham surgery or TAC 8 weeks post surgery. (A) Ctrl sham, n = 7; ctrl TAC, n = 9; CM-FGF21^−/− TAC, n = 7. (B) Ctrl sham, n = 6; ctrl TAC, n = 6; CM-FGF21^−/− TAC, n = 6. (C) Ctrl sham, n = 9; ctrl TAC, n = 12; CM-FGF21^−/− TAC, n = 11. (D–J) Ctrl sham, n = 6; ctrl TAC, n = 6; CM-FGF21^−/− TAC, n = 6. (K) Ctrl sham, n = 3–7; ctrl TAC, n = 6–11; CM-FGF21^−/− TAC, n = 5–8. (L) Ctrl sham, n = 6; ctrl TAC, n = 8; CM-FGF21^−/− TAC, n = 7. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using by one-way ANOVA with Tukey multiple comparison test. Inhibition of FGF21 signaling reversed pro-hypertrophic myocardial transcriptome, metabolome, and lipidome profiles To identify common pathways involved in the cardioprotective effects of FGF21 suppression, we compared the cardiac transcriptome (RNA sequencing [RNA-seq]), lipidome, and metabolome profiles between HEP-FGF21^−/− and CM-FGF21^−/− mice with TAC against control mice with TAC. The HEP-FGF21^−/− TAC mice showed 721 differentially expressed genes (403 upregulated and 318 downregulated) ([269]Figures 5A–5C; [270]Figure S9A), while CM-FGF21^−/− TAC mice had 1,155 (519 upregulated and 636 downregulated) ([271]Figures 5B–5D and [272]S9B) compared to control mice with TAC. Among these, 406 genes were commonly altered in both HEP-FGF21^−/− and CM-FGF21^−/− mice (202 upregulated and 204 downregulated) ([273]Figures 5B and 5C; [274]Tables S8A and S8B). KEGG pathway analysis indicated that the ECM receptor interaction-related gene expression program was suppressed in both genetic models ([275]Figures 5E and [276]S10A–S10D). However, PI3K-AKT, hypertrophic, and dilated cardiomyopathy pathways were induced in controls mice with TAC, surprisingly not reversed in HEP-FGF21^−/− mice ([277]Figures S11A and S11B), but were attenuated in CM-FGF21^−/− mice ([278]Figures S11C and S11D). Notably, CM-FGF21^−/− mice exhibited increased BCAA catabolism and fatty acid degradation-related gene expression ([279]Figures S11C and S11D), suggesting restoration of metabolic pathways. Figure 5. [280]Figure 5 [281]Open in a new tab The anti-hypertrophic effects of hepatocyte or CM FGF21 suppression share partial overlap in transcriptome, metabolome, and lipidome profile changes; oxytocin signaling is a common activated pathway (A–F) Hierarchical clustering (A and D), overlapping gene expression changes (B and C), and KEGG pathway analysis (E and F) depicting upregulation (A, B, D, and F) or downregulation (A, C, D, and E) in hearts obtained from HEP-FGF21^−/− or CM-FGF21^−/− mice subjected to TAC and compared to control mice subjected to TAC. (G) Hypothalamic oxytocin (OXT) mRNA levels in control mice and HEP-FGF21^−/− mice subjected to sham surgery or TAC for 3 days, 2 weeks, and 8 weeks post surgery. (H) Cardiac OXTR mRNA levels in C57BL/6 mice subjected to sham or TAC for 3 days, 2 weeks, 4 weeks, and 8 weeks. (I and J) Cardiac OXTR and β-actin immunoblotting (I) and quantification (J) in control mice subjected to sham or TAC surgery and HEP-FGF21^−/− mice after TAC 8 weeks post surgery. (K and L) Phalloidin and DAPI staining (scale bar, 100 μm) (K) and quantification of cell size (L) of adult CM treated with rm-FGF21, combination of rm-FGF21, and increasing doses of oxytocin or control vehicle for 24 h. (M) Summary of metabolomic and lipidomic changes depicting upregulation or downregulation in hearts obtained from HEP-FGF21^−/− or CM-FGF21^−/− mice subjected to TAC and compared to control mice subjected to TAC. (A–F) Ctrl TAC, n = 4–5; HEP-FGF21^−/− TAC, n = 5; CM-FGF21^−/−, n = 5. (G) Sham, n = 7; 3 days TAC, n = 6; 2 weeks TAC, n = 6; 8 weeks TAC, n = 6; HEP-FGF21^−/− 8 weeks TAC, n = 5. (H) Sham, n = 6; 3 days TAC, n = 12; 2 weeks TAC, n = 11; 4 weeks TAC, n = 12; 8 weeks TAC, n = 6. (I, J) Ctrl sham, n = 6; ctrl TAC, n = 11; HEP-FGF21^−/− TAC, n = 10. (K, L) Vehicle, n = 5; rmFGF21, n = 6; rmFGF21 OXT 10 nM, n = 4; rmFGF21 OXT 100 nM, n = 5. (M) Ctrl TAC, n = 4–5; HEP-FGF21^−/− TAC, n = 5; CM-FGF21^−/−, n = 5. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using one-way ANOVA with Tukey multiple comparison test or Student’s t test when applicable. Metabolomic analysis revealed higher levels of pyruvate and fumarate (Krebs cycle metabolite) in HEP-FGF21^−/− ([282]Figure 5M; [283]Table S5D) and CM-FGF21^−/− mice ([284]Figure 5M; [285]Table S5E) 8 weeks post TAC. Moreover, HEP-FGF21^−/− mice have higher citrate, NAD^+, and ATP levels, suggesting improvement in cardiac energetics ([286]Figure 5M; [287]Table S5D). Cardiac lipidome profiling showed that the increase in various phospholipids (PC, PE, and PS) observed in control mice with TAC was mostly reversed in HEP-FGF21^−/− ([288]Figure 5M; [289]Table S6D) and CM-FGF21^−/− ([290]Figure 5M; [291]Table S6E) mice, particularly for PC 17:1/18:2, PE 16:1/18:1, PE 18:0/18:2, PE 22:0/20:4, PE 22:0/22:6, and PS 18:4/22:4. This suggests that FGF21 deletion in hepatocytes or CM attenuates a subset of gene expression associated with cardiomyopathy and lowers phospholipid biosynthesis, likely due to the reversal of the hypertrophic program. The cardioprotective effect of FGF21 inhibition is associated with increased oxytocin signaling in the heart Strikingly, the expression of genes that encode proteins involved in the cardioprotective oxytocin signaling was increased in both HEP-FGF21^−/− and CM-FGF21^−/− mice ([292]Figure 5F). Interestingly, components of this pathway had the highest representation among the upregulated pathways in CM-FGF21^−/− mice with TAC compared to control mice with TAC ([293]Figures S11C and S11D). As protection from oxytocin signaling has been attributed to either a cardiac autocrine pathway or endocrine effects of oxytocin that is produced and released by the hypothalamus, we measured oxytocin expression in both organs during the progression of LVH development. Our analysis revealed that cardiac oxytocin expression does not change at any time point in mice with TAC ([294]Figure S12A) or in hearts of humans with hypertension ([295]Figure S12B), thus excluding potential autocrine effects. Conversely, oxytocin expression in the hypothalamus is significantly suppressed upon maximal release of FGF21 in the circulation (3 days post-TAC) and remains at low levels until 8 weeks post TAC ([296]Figure 5G). Notably, oxytocin expression in the hypothalamus is not suppressed in HEP-FGF21^−/− mice that have low levels of systemic FGF21 and are protected from PO-induced LVH ([297]Figure 5G). On the cardiac side, the levels of oxytocin receptor (OXT-R) mRNA in wild-type hearts are increased at the early stage of TAC (3 days), prior to the development of pathological hypertrophy, and gradually return to normal levels by 8 weeks post TAC ([298]Figure 5H). Measurement of OXT-R protein levels in the hearts of wild-type and HEP-FGF21^−/− mice 8 weeks post TAC revealed a 55% suppression in wild-type mice, which did not occur in the protected HEP-FGF21^−/− mice ([299]Figures 5I and 5J). Thus, inhibition of hepatic FGF21 expression in mice with TAC maintains normal levels of oxytocin expression in the hypothalamus and OXT-R in the heart. To investigate if oxytocin can reverse CM hypertrophy, we treated primary CMs isolated from adult mice with a combination of FGF21 and increasing levels of oxytocin. Analysis of the CM area showed that oxytocin attenuated the pro-hypertrophic effect of FGF21 ([300]Figures 5K and 5L). Thus, suppression of oxytocin signaling can exacerbate the effect of FGF21 in promoting LVH. The cardioprotective effect of hepatic FGF21 deletion is abolished upon CM FGF21 activation To further explore if CMs FGF21 per se is a main cause for the development of pathological hypertrophy with PO, we induced CM FGF21 overexpression in the HEP-FGF21^−/− and FGF21^fl/fl mice using a cardiotropic Myo-AAV9^1A-cardiac troponin T (cTnT)-FGF21[301]^33 vector that encodes for mouse FGF21 under the control of the CM-specific cTnT promoter (Myo-AAV9^1A-cTnT-FGF21). Four weeks post AAV injection, both groups underwent TAC and were monitored for 8 weeks. Control HEP-FGF21^−/− and FGF21^fl/fl mice were injected with empty Myo-AAV9^1A-cTnT, and TAC was applied. FGF21^fl/fl mice that were injected with empty Myo-AAV9^1A-cTnT and subjected to sham surgery were also used as an additional control group. We first confirmed that injection of Myo-AAV9^1A-cTnT-FGF21 increased cardiac FGF21 mRNA ([302]Figure 6A) and protein levels ([303]Figures 6B and 6C) compared to FGF21^fl/fl mice injected with empty Myo-AAV9^1A-cTnT. As expected, control FGF21^fl/fl mice that were injected with Myo-AAV9^1A-cTnT-FGF21 and subjected to TAC developed cardiac hypertrophy ([304]Figure 6D) and cardiac dysfunction ([305]Figures 6E and 6F; [306]Table S9) compared to HEP-FGF21^−/− mice injected with control virus and subjected to TAC or the FGF21^fl/fl mice injected with control Myo-AAV9^1A-cTnT and subjected to sham surgery. Strikingly, however, the protective effect of hepatocyte Fgf21 deletion against TAC-induced hypertrophy ([307]Figure 6D) and cardiac dysfunction ([308]Figures 6E and 6F; [309]Table S9) was abolished in HEP-FGF21^−/− mice, in which cardiac FGF21 expression was stimulated with Myo-AAV9^1A-cTnT-FGF21. This was accompanied by increased HF markers ([310]Figures 6G and 6H), CM enlargement ([311]Figures 6I and [312]S12C), increased myocardial fibrosis ([313]Figures 6J and [314]S12D), and higher fibrosis-related gene expression ([315]Figures S12E and S12F). Thus, CM FGF21 expression alone drives PO-induced hypertrophy, even without hepatic FGF21 signaling. Figure 6. [316]Figure 6 [317]Open in a new tab AAV-mediated overexpression of cardiac FGF21 negates the cardioprotective effect of hepatocyte-specific FGF21 ablation in PO Cardiac FGF21 mRNA (A), cardiac FGF21 immunoblotting (B), protein quantification (C), heart weight normalized to tibia length (D), representative short-axis M-mode images of the left ventricle (E), fractional shortening (F), cardiac BNP mRNA (G), cardiac β-MHC mRNA (H), WGA staining of the hearts (I; scale bar, 50 μm), and PSR staining of the hearts (J; scale bar, 100 μm) of control (floxed or Alb-Cre^+/−) or HEP-FGF21^−/− mice that were administered retro-orbitally either MyoAAV9^1A-ctrl or MyoAAV9^1A-FGF21 and subjected to either sham surgery or TAC for 8 weeks. (A) Myo AAV-Ctrl sham, n = 7; Myo AAV-Ctrl TAC, n = 7; Myo AAV-FGF21 TAC, n = 8; HEP-FGF21^−/− Myo AAV-Ctrl TAC, n = 6; HEP-FGF21^−/− Myo AAV-FGF21 TAC, n = 6. (B and C) Myo AAV-Ctrl sham, n = 4; Myo AAV-Ctrl TAC, n = 6; Myo AAV-FGF21 TAC, n = 6; HEP-FGF21^−/− Myo AAV-Ctrl TAC, n = 6; HEP-FGF21^−/− Myo AAV-FGF21 TAC, n = 6. (D) n = 6. (E and F) n = 6. (G and H) Myo AAV-Ctrl sham, n = 7; Myo AAV-Ctrl TAC, n = 9; Myo AAV-FGF21 TAC, n = 9; HEP-FGF21^−/− Myo AAV-Ctrl TAC, n = 6; HEP-FGF21^−/− Myo AAV-FGF21 TAC, n = 6. (I and J) n = 6. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using one-way ANOVA with Tukey multiple comparison test. ASO-mediated inhibition of FGF21 is cardioprotective against TAC-induced HF To investigate the translational potential of FGF21 inhibition, we developed anti-sense oligonucleotides (ASO-FGF21) targeting FGF21. We screened 10 candidate ASOs ([318]Table S10) (designed against mouse FGF21) in vitro using HL-1 cells treated with 250 or 500 nM ASOs for 48 h ([319]Figure S13A). ASO-FGF21-[2] (5′-GGUGACGGGGGAAAGUAGGU 3′) showed the highest suppression (70% at 250 nM) and was validated in HL-1 cells ([320]Figure S13B). The selected ASO-FGF21 and ASO-scr were chemically modified for in vivo stability (central gap segment comprising ten 2′-deoxynucleosides and flanked on the 5′ and 3′ wings by five 2′-O-methoxyethyl-modified nucleosides) and administered to wild-type mice via retroorbital injection (10 mg/kg body weight [BW]) weekly for 4 weeks. This resulted in significant suppression of cardiac (88%), hepatic (94%), and plasma (48%) FGF21 levels ([321]Figures S13C–S13E). The most effective ASO-FGF21 was tested in vivo in wild-type mice with TAC or control surgery. For a group of mice, the ASO-FGF21 treatment (10 mg/kg BW weekly injections) started the next day after TAC and lasted 8 weeks ([322]Figure 7A). Another group of wild-type mice with TAC was injected with control ASO-scr for the first 2 weeks post TAC. At the 2-week time point, when pathological hypertrophy is observed ([323]Figure 1D), we performed weekly injections with ASO-FGF21 for 6 more weeks ([324]Figure 7A). Control wild-type mice with either sham surgery or TAC were treated with ASO-scr for 8 weeks ([325]Figure 7A). τhe ASO-FGF21 treatment lowered the expression of FGF21 in the liver (69% for 0–8 weeks and 63% for 2–8 weeks, [326]Figure 7B), heart (49% for 0–8 weeks and 48% for 2–8 weeks, [327]Figure 7C), and WAT (38% for 0–8 weeks and 41% for 2–8 weeks, [328]Figure 7D). In accordance with the genetic FGF21 loss-of-function models, ASO-FGF21 alleviated cardiac hypertrophy ([329]Figure 7E) and improved cardiac function ([330]Figures 7F and 7G; [331]Table S11) in both treatment protocols (0–8 weeks and 2–8 weeks). Accordingly, CM enlargement ([332]Figures 7H and 7I), myocardial fibrosis ([333]Figures 7J and 7K), cardiac fibrosis-related ([334]Figures S14A–S14C), and inflammation-related gene expression ([335]Figures S14D–S14F) were attenuated. Furthermore, the ASO-FGF21 treatment reduced the expression levels of HF markers BNP ([336]Figure S14G) and β-MHC ([337]Figure S14H). In conclusion, systemic or CM-specific inhibition of FGF21 holds therapeutic value for LVH and cardiac dysfunction that are induced by increased afterload. Figure 7. [338]Figure 7 [339]Open in a new tab Antisense oligonucleotide-mediated inhibition of FGF21 holds therapeutic potential against LVH from increased afterload (A) Outline of the treatment of C57BL/6 mice that were subjected to control sham surgery or TAC with control ASO-scr, ASO-FGF21, or sequential combination of both. (B–K) Hepatic (B), cardiac (C), and WAT FGF21 mRNA (D), heart weight normalized to tibia length (E), representative short-axis M-mode images of the left ventricle (F), measurements of fractional shortening (G), WGA staining (H; scale bar, 50 μm) and quantification of cell size (I), and PSR staining (J; scale bar, 100 μm) and quantification of the fibrotic tissue area (K) in C57BL/6 mice subjected to sham surgery or TAC followed by treatment with control ASO-scr for 8 weeks, ASO-FGF21 for 8 weeks, or sequential treatment with ASO-scr for 2 weeks and ASO-FGF21 for 6 additional weeks. (B–D) Sham ASO Scr, n = 5–6; TAC ASO Scr, n = 6–7; TAC ASO FGF21 (0–8 weeks), n = 5–6; TAC ASO FGF21 (2–8 weeks), n = 6. (E–K) Sham ASO Scr, n = 6; TAC ASO Scr, n = 6; TAC ASO FGF21 (0–8 weeks), n = 5–6; TAC ASO FGF21 (2–8 weeks), n = 6. Error bar represented ±SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; analyzed using one-way ANOVA with Tukey multiple comparison test. “n” refers to biological replicates. For all quantitative reverse-transcription PCR (RT-qPCR), echocardiography, and plasma FGF21 analyses, each biological replicate was analyzed in two technical replicates. For micrographs, each biological replicate was analyzed in three technical replicates. For immunoblotting images, each biological replicate corresponds to one technical replicate. Discussion The heart’s primary function is to pump blood efficiently, delivering oxygen and nutrients to organs. In response to increased preload or afterload, CMs and the left ventricle enlarge, initially enhancing contractility in a process called physiological hypertrophy, seen in exercise. However, prolonged thickening leads to pathological hypertrophy, often due to aortic stenosis or hypertension,[340]^2 and is characterized by fibrosis, inflammation, and increased risk of HF and mortality.[341]^3^,[342]^4 While several signaling pathways regulate hypertrophy, the systemic mechanisms driving cardiac hypertrophy remain incompletely understood. In myocardial samples from patients with heart failure with reduced ejection fraction (HFrEF),[343]^22 increased FGF21 staining and elevated serum FGF21 levels in LVH and hypertension ([344]Figures 1A and 1B) prompted us to investigate FGF21’s role in LVH. Our findings in a mouse model of PO suggest a pro-hypertrophic signaling pathway, which entails liver-heart crosstalk that is spearheaded by FGF21. Interestingly, hepatic FGF21 induces CM FGF21 expression, promoting LVH through an autocrine mechanism. FGF21 is an endocrine metabolic hormone induced by nutritional and physiological stress,[345]^5^,[346]^6^,[347]^13^,[348]^14^,[349]^15 primarily produced in the liver[350]^34 but also in the heart.[351]^12^,[352]^35 Plasma FGF21 levels are elevated in obese humans and animal models.[353]^36^,[354]^37^,[355]^38^,[356]^39^,[357]^40 Although this could be perceived as a component of a pathological mechanism, a study that suggested FGF21 resistance in obese mice concluded that FGF21 production in obesity is a protective mechanism.[358]^17^,[359]^18^,[360]^19^,[361]^20^,[362]^21^,[363]^41^,[ 364]^42 While its transient benefits are obvious, the long-term effects on obesity remain unclear,[365]^10^,[366]^11^,[367]^37^,[368]^41^,[369]^43^,[370]^44^,[37 1]^45^,[372]^46^,[373]^47 making its role in body weight regulation controversial. The role of FGF21 in cardiac physiology remains controversial. Our results show that the inhibition of hepatocyte FGF21 is cardioprotective against TAC-induced HF. Moreover, the inhibition of CM FGF21 is also protective despite high levels of hepatic FGF21 expression and serum FGF21 levels, pointing to a cardiotoxic role of FGF21. Accordingly, an FGF21 analog that has been used in humans increased systolic and diastolic blood pressure.[374]^48 However, some studies indicate that FGF21 may be cardioprotective in specific contexts, such as myocardial infarction,[375]^49 doxorubicin toxicity,[376]^50 and A[2A] receptor activation in hypertension.[377]^51 Global FGF21 deletion exacerbates isoproterenol-induced hypertrophy but had uncertain long-term effects.[378]^12 Similarly, global FGF21 deletion in hypertensive mice led to cardiac dysfunction and fibrosis,[379]^52 while liver/muscle FGF21 overexpression improved cardiac function, 2 weeks post ischemia but not beyond that time point.[380]^53 FGF21 treatment was also protective in isolated rat hearts during global ischemia.[381]^54 In diabetic mice and obese humans, exercise failed to improve heart function in FGF21^−/− mice, suggesting a protective role in diabetic cardiomyopathy.[382]^55 However, exercise-induced benefits were linked to lower plasma FGF21 levels. While FGF21 appears cardioprotective in the short term, its long-term effects remain uncertain. To this end, a human study found increased FGF21 staining in HFrEF heart biopsies,[383]^22 while higher plasma FGF21 levels correlate with diastolic dysfunction, mortality in HFpEF,[384]^23 or patients with cardiomyopathy secondary to persistent atrial fibrillation.[385]^29 A recent human study also revealed a strong association of FGF21 with hypertrophic cardiomyopathy.[386]^56 Hypertension studies showed higher circulating FGF21 levels in mice,[387]^52 as well as in humans,[388]^24 which we also observed ([389]Figure 1). Elevated FGF21 levels were also observed in hypertension and a genetic hypertrophic cardiomyopathy model.[390]^57 Notably, global FGF21 deletion protected mice from pregnancy-induced cardiomyopathy.[391]^58 These findings suggest that prolonged FGF21 signaling may aggravate cardiac dysfunction at least in certain cardiomyopathies. To address discrepancies between clinical and preclinical studies using global FGF21^−/− mice, we investigated tissue-specific FGF21 knockout models. Our findings show that CM FGF21 induction is crucial for pathological hypertrophy, while a previous study found that constitutive CM FGF21 expression did not cause hypertrophy but reduced fatty acid oxidation gene expression,[392]^47 resembling PO-induced LVH.[393]^59 To test if cardiac stress was needed, we combined AAV-mediated FGF21 transfer with TAC in HEP-FGF21^−/− mice, which were protected from LVH. This confirmed that CM FGF21 induction negates the protective effects of hepatocyte FGF21 deletion against PO-induced LVH. In addition to other studies and ours, which show that chronic FGF21 upregulation aggravates cardiac dysfunction, we also identified a time lag of approximately 2 weeks between the increase in hepatic and plasma FGF21 levels and the stimulation of CM FGF21 expression that leads to pathological hypertrophy and HF, accompanied by fibrosis and inflammation. We and other groups have shown that suppression of fibroblast activation prevents TAC-induced cardiac hypertrophy.[394]^31^,[395]^60^,[396]^61 Thus, cardiac hypertrophy with PO seems to require both fibroblast activation and induction of CM FGF21 expression. It remains to be elucidated whether fibroblast activation precedes or follows CM FGF21 upregulation. Moreover, as it has been shown that non-cardiac organs or cells may mediate either the effects of FGF21 or cardiac hypertrophy, such as the central nervous system[397]^62 or inflammatory cells,[398]^63 the involvement of other systems in modulating the pro-hypertrophic effect of FGF21 remains to be explored. One of the pathways that were induced with the protective inhibition of either hepatic or CM FGF21 was oxytocin signaling. Oxytocin is a neuropeptide that can be produced either by the hypothalamus or the heart[399]^64 and has been associated with cardiac protection in various types of cardiomyopathy.[400]^65 Specifically, oxytocin prevents CM hypertrophy, as shown by others[401]^66 and our new data, and LVH and fibrosis in ovariectomized rats.[402]^67 It also attenuates parasympathetic activation that protects from myocardial ischemia-induced complications,[403]^68 oxidative stress,[404]^69 inflammation,[405]^70 and atrial natriuretic peptide release that lowers cardiac afterload.[406]^71^,[407]^72 Oxytocin exerts its signaling effects via binding on OXT-R, which is a G[q]-protein-coupled receptor. OXT-R is present in many tissues including the heart of humans and rodents.[408]^71^,[409]^72^,[410]^73 In our study, oxytocin expression was not affected in the heart. Although FGF21 increases oxytocin expression in hypothalamus,[411]^74 we show that prolonged systemic FGF21 upregulation leads to its suppression, likely via a negative feedback loop mechanism. Our in vitro studies demonstrate that exposing CMs to oxytocin prevents FGF21-induced enlargement of cardiac cell size ([412]Figures 5K and 5L). This finding is aligned with our in vivo RNA-seq analysis, which shows that oxytocin signaling components are restored following the genetic deletion of FGF21 in either the liver or heart during PO. These results suggest that FGF21 signaling reduces oxytocin signaling in the heart. Future studies should explore whether competition between FGF21 and oxytocin signaling contributes to the progression of pathological LVH. The importance of identifying the potential involvement of FGF21 in LVH is highlighted by the use of recombinant FGF21, stable FGF21 analogs, and receptor agonists in preclinical[413]^17^,[414]^19^,[415]^20^,[416]^75^,[417]^76^,[418]^77^, [419]^78^,[420]^79^,[421]^80^,[422]^81^,[423]^82^,[424]^83^,[425]^84^,[ 426]^85 and phase 1 and 2 clinical studies[427]^17^,[428]^18^,[429]^21^,[430]^86^,[431]^87 for the treatment of obesity, insulin resistance, hyperglycemia, severe hypertriglyceridemia, atherosclerosis, and hepatic steatosis. Our findings suggest that such therapies need to be applied with extra caution to people at increased risk for HF. Moreover, more thorough phenotyping of clinical samples in the future will provide evidence for definitive association between FGF21 levels and clinical variables representing disease severity. This may identify FGF21 as a strong biomarker for risk stratification and an objective target for potential treatment of LVH. Conclusion Our study reveals that PO induces LVH through an endocrine-autocrine hepato-cardiac mechanism, with FGF21 as the key mediator. Liver-derived FGF21 responds early to cardiac stress, triggering CM FGF21 expression in an autocrine manner. Additionally, FGF21 may impair cardioprotective signaling by reducing hypothalamic oxytocin expression. Targeting FGF21 signaling could offer a therapeutic approach for LVH caused by increased afterload. Limitations of the study This preclinical study identifies FGF21’s role in LVH in pressure-overloaded mice. While serum and myocardial FGF21 were elevated in patients with LVH (serum) or hypertension (serum and hypertension), their metabolic comorbidities may influence systemic FGF21 levels. Complete FGF21 suppression could have metabolic side effects, making partial inhibition, as seen with our ASO-FGF21 and tissue-specific knockouts, a preferable approach. Efforts to develop stable FGF21 analogs and receptor agonists are ongoing, with clinical trials for metabolic diseases. Future studies should evaluate prolonged FGF21 treatment in type 2 diabetic mice and its effects on the myocardium. Our study focused on cardiac hypertrophy due to increased afterload and did not analyze early transcriptomic, lipidomic, or metabolomic changes in the HEP-FGF21^−/− and CM-FGF21^−/− mice before 8 weeks post TAC. Since we used young mice, caution is needed when extrapolating results to older patients with hypertrophy and HF. To secure sufficient biological replicates in our experimental groups (at least 6/group), some of our control mouse cohorts include a combined population of floxed mice and tissue-specific (either hepatic or cardiac depending on the experiment) Cre-expressing mice. However, our pilot studies ([432]Figures S3D–S3G) showed that Cre-expressing and non-expressing mice exhibit similar phenotypes. Resource availability Lead contact Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Konstantinos Drosatos (k.drosatos@uc.edu). Materials availability All unique reagents generated in this study are available from the [433]lead contact with a completed materials transfer agreement if required by the policy of the University of Cincinnati. The plasmid generated by the group of Jeffery D. Molkentin for the MyoAAV9^1A-cTnT-FGF21 is available upon request to the [434]lead contact with a completed materials transfer agreement. Data and code availability * • Raw and processed data from RNA-seq analysis of mouse hearts have been submitted to Gene Expression Omnibus (GEO).[435]^88 Bioproject accession number: [436]PRJNA1106030; series [437]GSE266368 , [438]GSE266957, and [439]GSE267618. * • Raw and processed data from metabolomic (NMR) and lipidomic (LC-MS/MS) analyses of mouse hearts have been submitted to Metabolomics Workbench,[440]^89 Project ID: [441]PR002329; Study IDs: ST003743 and ST003851. * • This paper does not report the original code. * • Any additional information required to reanalyze the data reported in this paper is available from the [442]lead contact upon request. Acknowledgments