Role Of Farnesylpyrophosphate Synthase In Angiotensin Ⅱ-induced Cardiac Hypertrophy

Part 1 Inhibition of farnesylpyrophosphate synthase prevents angiotensin II-induced hypertrophic responses in rat neonatal cardiomyocytes:involvement of the RhoA/Rho kinase pathwayBackground:Cardiac hypertophy can be induced by humal factors such as angiotensinⅡ(AngⅡ). Various pathways are involved in AngⅡ-induced hypertrophic response of cardiomyocytes including the RhoA/ROCK signaling.Experimental study has shown that alendronate inhibits farnesylpyrophosphate (FPP) synthase, a key enzyme in the mevalonate pathway, through inhibition of isoprenylation including farnesylation and geranylgeranylation with consecutively decreases of the formation of isoprenoid lipids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The latter one is essential for geranylgeranylation and activation of RhoA. Aim:We undertook our research to explore whether inhibition of FPP synthase by alendronate could interfere with the hypertrophic responses induced by AngⅡin cultured neonatal ventricular myocytes, and whether it involves RhoA/ROCK pathway.Methods:Cardiomyocyte were in serum-free medium (SFM) for 24 h before incubation with various agents with indicated time. Hypertrophy markers including cell surface area and protein content were assayed while gene expression of BNP was measured by quantitative real-time polymerase chain reaction (qRT-PCR).(1) Myocytes were pre-incubated with 3-30μM alendronate alone or with AngⅡ(1μM) for 48 h.(2) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH in the addition of AngⅡ(1μM) for 48 h.(3) Myocytes were pre-incubated with alendronate, GGTI-286, C3 exoenzyme or Y-27632 in the addition of AngⅡ(1μM) for 48 h(4) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH for 48 h before incubation with AngⅡfor 15 min. Pull down assay and western blot analysis for RhoA activity and expression were performed.Results:(1) Alendronate at 3 to 30μM prevented hypertrophy responses induced by AngⅡ(1μM).(2) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate in Ang II-cultured neonatal cardiomyocytes hypertrophy were partially reversed by comparative geranylgeranyol (GGOH).(3) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate could be mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho or Y-27632, an inhibitor of ROCK.(4) Pull-down assay showed alendronate reduced-active RhoA by Ang II was also partially antagonized by GGOH.GGTI-286 also abolished the activated RhoA by AngⅡ.Conlusions:This study reveals the inhibition of FPP synthase by alendronte reduces RhoA activation by diminishing geranylgeranylation which prevents AngⅡ-induced hypertrophic response of neonatal cardiomyocytes. Part 2 Konckdown of farnesylpyrophosphate synthase by RNA interference prevents angiotensin II-induced cardiac hypertrophyBackground:Our first part experimental research suggests inhibition of farnesylpyrophosphate synthase (FPPS) enzyme could reverse cardiac hypertrophy induced by angiotensinⅡ(AngⅡ), which indicates FPPS gene as the therapeutic target involved in AngⅡ-induced cardiac hypertrophy.The data of our previous RNA array study reported the up-regulation of FPPS mRNA in hypertrophy myocardium of 18-week spontaneously hypertensive rats (SHR) associated with local activated renin-angiotensin system (RAS) and higher AngⅡcontent than the age-matched Wistar-Kyoto rats (WKY).Collectively, this evidence strongly suggests the probable essential role of FPPS in the development of cardiac hypertrophy by Ang II.Aim:The aim of our study was designed to investigate whether the mRNA and protein expression of FPPS are increased in AngⅡ-mediated cardiac hypertrophy, then whether silencing FPPS modulates these hypertrophic responses, and furthermore studied FPPS-related signaling.Methods:(1) FPPS expression was measured in cardiac hypertrophy models by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot.(2) The four different sequences of short interference RNA duplexes (siRNAs) targeting the rat FPPS gene were constructed into shRNA vectors and the FPPS fusion protein was generated by insertion of rat FPPS cDNA into the pEGFP-C1 vector. HEK293 T cells were co-transfected with the rat FPPS fusion protein plus different shRNAs against FPPS or scrambled shRNA using lipofectamine 2000 and then the most active shRNA against rat FPPS was established by observation of GFP expression by immunofluorescence and western blot.(3) The most active FPPS siRNA was established, synthesized and then incorporated into a lentiviral vector with scrambled shRNA control for lentivirus production in vitro and in vivo gene transfer experiment. Hypertrophy makers in vitro include cell surface area and the mRNA expression of P-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP) by qRT-PCR. Hypertrophic responses in vivo indexed by heart weight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW) and echocardiography were measured as well as expression ofβ-MHC and BNP mRNA by qRT-PCR.(4) The involvement of FPPS in Ang II-activated hypertrophic responses was furthered by investigation of downstream signaling in cultured cardiomyocytes. Pull down assay and western blot analysis for RhoA activity and expression were performed. RhoA immunofluorescence confocal microscopy was analysed for RhoA translocation. Besides, the activity of p-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) was performed by western blot analysis.Results:(1) FPPS expression was elevated both in cultured neonatal cardiomyocytes (NCMs) following AngⅡ(1μM) treatment and in the hypertrophic myocardium of 18-week-old spontaneously hypertensive rats (SHRs).(2) FPPS shRNA lentivirus was constructed successfully. FPPS silencing in NCMs completely inhibited the hypertrophy marker genes ofβ-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP), as well as cell surface area. In vivo gene transfer also attenuated hypertrophic responses as indexed by left ventricular weight/body weight, heart weight/body weight as well as expression ofβ-MHC and BNP mRNA in SHRs. Echocardiography also showed the partial attenuation of interventricular septum wall thickness at diastolic phase (IVSd) and improvement of fraction shortening (FS) and ejection fraction (EF) in SHR silencing group.(2) FPPS knockdown prevented elevated RhoA activity as well as RhoA translocation to plasma membrane in incubation with AngⅡcompared with non-silenced controls in NCMs. Similarly, increased-phosphorylation of P-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by AngⅡwas attenuated.Conlusions:In conclusion, the expression of FPPS was elevated in Ang II-mediated cardiac hypertrophy and FPPS with RhoA-associated P38 and JNK MAPK signaling might play an important role in AngⅡ-induced cardiac hypertrophy. Part 3 Alendronate modulates angiotensinⅡ-induced neonatal cardiac fibroblast proliferation differentiation and collagen production in vitroBackground:Nitrogen-containing bisphosphonates (N-BPs), are extensively used in bone-related diseases and cancers. Our first part experimental research suggests alendronate could modulate cardiac hypertrophy induced by angiotensinⅡ(AngⅡ). Previous study demonstrated alendronate has beneficial effects on cardiac fibrosis in spontaneously hypertensive rats.Aims:This study was designed to examine whether pretreatment with alendronate could modulate Ang II activated early events of cardiac fibrosis in vitro including the increase in cell proliferation, differentiation of neonatal cardiac fibroblasts, and collagen production.Methods:Cardiac fibroblasts were prepared from 1 to 2-day-old Wistar rats. The proliferation of cardiac fibroblasts was determined by water-soluble tetrazolium (WST)-1 assay using Cell Counting Kit (CCK-8) and the differentiation marker of cardiac fibroblasts a-smooth muscle actin (a-SMA) was measured by western blot. Collagen production was assayed by the mRNA expression of collagen type II and type II using the real-time polymerase chain reaction (qRT-PCR) method, as well as by the analysis of hydroxyproline levels. Additionally transforming growth factor (TGF)-β1 protein production by cultured neonatal cardiac fibroblasts as well as P38 mitogen-activated protein kinase (MAPK) activity was evaluated by enzyme linked immunosorbent (ELISA) and western blot respectively.Results:Alendronate inhibited the proliferation and differentiation of cardiac fibroblasts as well as prevented collagen production by Ang II in vitro. Furthermore, the TGF-β1 protein expression and secretion by cardiac fibroblasts as well as activated P38 MAPK by AngⅡwere all reversed by alendronate.Conclusion:The current study demonstrates the inhibitory role of alendronate on Ang II-induced increase in cell proliferation, differentiation of neonatal cardiac fibroblasts and collagen production. The effect is possibly mediated by lowering the TGF-β1 levels and inactivation of P38 MAPK signaling.