Role Of Corin In Mouse Models Of Heart Failure

Chapter 1:Therapeutic efficacy of recombinant soluble corin in wild-type mouse models of heart failureObjective:Atrial and B-type natriuretic peptides(ANP and BNP,respectively)are key hormones of the cardiac endocrine mechanism.These hormones increase vasodilation,natriuresis and diuresis,thereby regulating cardiovascular homeostasis.ANP-mediated signaling also serves as an anti-hypertrophic and anti-inflammatory mechanism in the heart.In cells,the natriuretic peptides are produced as inactive pro-forms,i.e.pro-ANP and pro-BNP.Upon secretion,pro-ANP and pro-BNP are cleaved by proteases to become active ANP and BNP.In patients with heart failure(HF),circulating pro-ANP and pro-BNP levels are highly elevated,suggesting an underlying rate-limiting step in natriuretic peptide processing in failing hearts.Corin is a transmembrane protease that activates the natriuretic peptides in the heart.Impaired corin expression and function are associated with HF.Low levels of circulating corin have been reported in HF patients with poor clinical outcomes.These results suggest that corin is a rate-limiting enzyme in processing natriuretic peptides in failing hearts.In this chapter,we examine if treatment of recombinant soluble corin(sCorin)improves cardiac function in two models of HF in wild-type(WT)mice.Methods:1.Male WT mice(10-12 weeks old)were used in two independent models of HF induced by myocardial infarction(MI)and transverse aortic constriction(TAC),respectively.2.Recombinant sCorin(3 mg/kg)or an equal volume of vehicle control was injected intraperitoneally(i.p.)(daily for 3-7 weeks),starting at one week post-surgery.3.Cardiac function was assessed by echocardiography before and after the surgery.4.At 4 weeks post-MI or 8 weeks post-TAC surgery,heart weight(HW)and lung weight(LW),normalized to body weight(BW)or tibia length(TL),were analyzed.Cardiac hypertrophy and pulmonary congestion were also assessed.5.Heart tissue sections were stained with hematoxylin and eosin(H&E),wheat germ agglutinin(WGA),Masson and Sirius Red to evaluate cardiac morphology,cardiac myocyte size and tissue fibrosis.6.At 4 weeks post-MI or 8 weeks post-TAC surgery,ELISA was used to measure plasma levels of ANP,BNP,N-terminal(NT)-pro-ANP,cGMP,angiotensin II and aldosterone.Results:1.In the HF model induced by left coronary artery ligation,mice of the MI group had decreased cardiac function,less viable myocytes and more scar tissues,fibrosis and pulmonary congestion,compared with the sham control group at one week post-MI.2.At 8 weeks post-TAC,the mice had reduced cardiac function and increased cardiac hypertrophy,as indicated by higher HW/BW ratios,compared with those in the sham controls.3.After 2 weeks(MI)or 5 weeks(TAC)of treatment with sCorin,the mice had improved cardiac function,as indicated by increased values of ejection fraction(EF)and fractional shortening(FS).4.sCorin-treated mice had reduced HW and LW that were normalized to BW or TL,an indication of reduced cardiac hypertrophy and pulmonary congestion in these mice.5.Histological analyses indicated decreased tissue fibrosis and reduced cardiomyocyte diameters in sCorin-treated mice,compared to those in vehicle-treated control mice.6.sCorin treatment increased ANP,BNP,and cGMP levels and decreased NT-pro-ANP,angiotensin Ⅱ,and aldosterone levels in plasma samples.Levels of cGMP in left ventricular(LV)tissues were also higher in sCorin-treated mice than vehicle-treated mice.Conclusions:We show that in WT mice sCorin treatment enhanced natriuretic peptide processing,suppressed the renin-angiotensin-aldosterone system(RAAS),and improved cardiac morphology and function in HF models induced by MI and TAC,respectively.Chapter 2:Cardiac function in corin knockout mice and the effects of sCorin in TAC-induced HF modelObjective:To date,CORIN gene mutations or variants have been reported in patients with hypertension and heart disease in different populations,suggesting that CORIN gene defects may impair corin expression and/or function,thereby contributing to hypertension and heart disease.It remains to be determined experimentally if corin deficiency directly causes cardiac dysfunction and if corin deficiency accelerates cardiac dysfunction under certain pathological conditions.Additionally,it is unclear if recombinant sCorin treatment could ameliorate cardiac dysfunction associated with corin deficiency.In this chapter,we used corin knockout(KO)mice as an animal model to examine the effect of corin deficiency on cardiac function and the effect of sCorin on cardiac morphology and function in corin KO mice subjected to TAC.Methods:1.Male WT and corin KO mice(6 and 12 months old)were examined with echocardiography to evaluate their cardiac function at different ages.2.Male WT and corin KO mice(10-12 weeks old)were tested for the HF model induced by TAC.Cardiac function changes in WT and corin KO mice after TAC were compared to assess whether corin deficiency accelerates cardiac dysfunction under pathological conditions.3.sCorin(3 mg/kg)or control vehicle was injected(i.p.daily for 7 weeks),starting at one week post-surgery.Cardiac function was assessed by echocardiography before and after the surgery.Heart and lung tissues were collected 8 weeks after surgery.4.At 8 weeks post-TAC surgery,HW and LW to BW or TL ratios were analyzed.Cardiac hypertrophy and pulmonary congestion were assessed.5.At 8 weeks post-TAC surgery,heart tissue sections were stained with H&E,WGA,Masson and/or Sirius Red to evaluate cardiac morphology,cardiomyocyte size,and tissue fibrosis.6.Expression levels of HF-related genes,including Myh7,Ctgf,Nppa and Nppb,in heart tissues were analyzed by qRT-PCR.7.At 8 weeks post-TAC surgery,plasma levels of cGMP,N-terminal(NT)-pro-ANP,angiotensin Ⅱ and aldosterone were examined with ELISA.Results:1.Compared with age-matched WT mice,corin KO mice had significantly lower EF values at 12 months,suggesting cardiac dysfunction at an old age.2.After the TAC treatment,values of EF decreased to 50%in 6.0±0.2 weeks in WT mice,whereas in 3.9±0.1 weeks in corin KO mice(p<0.05),suggesting that corin deficiency accelerated the HF development in mice.3.After 7 weeks of treatment with sCorin,corin KO mice subjected to TAC had improved cardiac function,as indicated by increased values of EF and FS.4.sCorin-treated corin KO mice had reduced cardiac hypertrophy and fibrosis,smaller cardiomyocyte diameters,and less pulmonary congestion,suggesting improved cardiac function in these mice.5.qRT-PCR analysis showed that the expression levels of HF-related genes,including Myh7,Ctgf,Nppa and Nppb,were significantly decreased in hearts from sCorin-treated corin KO mice.6.sCorin-treated corin KO mice had increased cGMP levels and decreased NT-pro-ANP,angiotensin Ⅱ and aldosterone levels in plasma samples collected at 8 weeks post-TAC.Conclusions:Corin KO mice had cardiac dysfunction at 12 months of age.When subjected to the TAC procedure,corin KO mice were more susceptible to the development of HF,indicating that corin deficiency accelerated the HF development in these mice.In the TAC model,corin KO mice treated with recombinant sCorin had increased cardiac function,improved cardiac morphology,less cardiac tissue fibrosis,and increased levels of plasma natriuretic peptide antigen and activity,compared to those in control mice.These results indicate that corin deficiency contributes to the pathogenesis of HF and that recombinant sCorin may be considered as a therapeutic approach to treat HF associated with corin deficiency.Chapter 3:Effects of Corin gene knockout on cardiomyocytes and cardiac injury responses in miceObjective:The Corin-ANP pathway is essential for normal blood pressure.ANP also has an anti-hypertrophic function in the heart,which is independent of its systemic blood pressure lowering effect.Both ANP and corin KO mice develop hypertension and cardiac hypertrophy.It remains to be determined if observed changes in the heart of corin KO mice were caused by elevated blood pressure or a local mechanism that is yet recognized.In principle,Corin defects could have a direct effect on cardiac structure and/or function in addition to its effects on systemic blood pressure.In this chapter,we examine effects of corin deficiency on cultured neonatal cardiomyocytes and cardiac responses to different injuries in corin KO mice.Methods:1.Cardiomyocytes from WT and corin KO neonatal mice were isolated and cultured.Morphology and spontaneous beating frequency of the cultured cells were analyzed.2.Male WT and corin KO mice(10-12 weeks old)were subjected to coronary artery ligation,and one-day survival rate was examined.Heart tissues were collected from WT and corin KO mice at the end of the experiment.3.Triphenyl tetrazolium chloride(TTC)staining and TNF-α immune staining were performed with the heart tissues to evaluate ischemic sizes and TNF-α protein expression levels.4.Expression levels of inflammatory cytokine genes,including TNF-α,IL-1β,IL-6 and IL-10,in the heart tissues were assessed by qRT-PCR.5.Hearts of male WT and corin KO mice were subjected to liquid nitrogen treatment and survival rates in the mice were analyzed.H&E-stained heart tissue sections were analyzed under a light microscope.6.Coronary arteries in WT and corin KO mice were examined with Evans blue staining and immune staining of α-smooth muscle actin(α-SMA)(vascular smooth muscle cell marker)to assess the size and vessel wall structure.Results:1.No significant differences were observed in cell number,morphology,and proliferation rate of cultured primary cardiomyocytes between WT and corin KO mice.Spontaneous beating frequencies of the cultured primary cardiomyocytes from WT and corin KO mice were also similar.2.Virtually all corin KO mice died within one day after coronary artery ligation,whereas most WT mice survived after the similar treatment.TTC staining showed no significant difference in ischemic area sizes between the two groups.Levels of inflammatory cytokine gene expression and TNF-α protein were also similar in the two groups.3.Both WT and corin KO mice survived after their hearts were subjected to liquid nitrogen injury and there was no difference in survival rate between the two groups.Histological analysis in H&E-stained heart sections did not find differences in morphology and cellular components.4.Analysis of coronary artery structures revealed that,compared to those in WT mice,corin KO mice had smaller coronary arteries with thinner vessel walls,which may contribute to the low survival rate in corin KO mice subjected to coronary artery ligation.Conclusions:Corin deficiency had no significant effects on the morphology,growth,and spontaneous beating frequency in cultured primary cardiomyocytes from neonatal mice.Compared to WT mice,corin KO mice had a higher mortality rate caused by coronary artery ligation.Morphological and histological analyses revealed a defect in coronary artery wall structure with a thinner layer of smooth muscle cells in corin KO mice.This defect may contribute to the susceptibility to acute cardiac ischemia-induced death in corin KO mice.