Prolylcarboxypeptidase Alleviates Hypertensive Cardiac Remodeling by Regulating Myocardial Tissue Angiotensin II

Abstract

Background Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1-7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro-X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results We established a CRISPR/CRISPR-associated protein 9-mediated global prolylcarboxylpeptidase-knockout and adeno-associated virus serotype 9-mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild-type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II-treated prolylcarboxylpeptidase-knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase-knockout hearts showed upregulated extracellular signal-regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno-associated virus serotype 9-mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase-knockout hearts alleviated angiotensin II-induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno-associated virus serotype 9-mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II-induced cardiac dysfunction. Conclusions Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II-induced hypertrophic remodeling by controlling myocardial angiotensin II levels.

Keywords: hypertension; hypertrophy; lysosome; prolylcarboxylpeptidase.

Figure 1. Alteration of cardiac prolylcarboxylpeptidase expression in response to angiotensin II stress.

A, While cardiac ACE2 expression was continuously downregulated, cardiac prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated (n=4 per group). One‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. Data are expressed as means±SEM. B, ACE2 expression was decreased after angiotensin II (100 nmol/L) treatments, whereas prolylcarboxylpeptidase was increased after 24 hours of angiotensin II treatment and then decreased in human induced pluripotent stem cell–derived cardiomyocytes. One‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. Data are expressed as means±SEM (n=4 per group). C, Prolylcarboxylpeptidase expression was downregulated in heart tissue samples of patients with CDM, compared with controls. Unpaired 2‐tailed Student's t‐test was used for analysis. Data are expressed as means±SEM (n=4 for control, n=8 for CDM). ACE2 indicates angiotensin‐converting enzyme 2; AngII, angiotensin II; A.U, Arbitrary unit; CDM, cardiomyopathy; and PRCP, prolylcarboxylpeptidase.

Figure 2. Prolylcarboxylpeptidase deficiency led to reduced cardiac function with augmented hypertrophic remodeling upon 4 weeks of angiotensin II infusion.

A, Diastolic and systolic blood pressure after 4 weeks of angiotensin II infusion. B, WGA staining of heart cross‐sections (scale bar: 50 μm). C, Left ventricle end‐diastolic posterior wall. D, Mitral Doppler showed the ratio of peak velocity blood flow from left ventricular relaxation in early diastole (the E wave) to peak velocity flow in late diastole caused by atrial contraction (the A wave) indicated worsen diastolic function in prolylcarboxylpeptidase‐knockout mice. E, Fractional shortening (%) was significantly decreased in prolylcarboxylpeptidase‐knockout mice. F, Masson's trichrome‐staining of cross‐sections showed significantly increased interstitial fibrosis in prolylcarboxylpeptidase‐knockout mice compared with the same cohort of control mice (scale bar: 30 μm), followed by quantification of fibrosis area against total tissue area (n=8 mice per group). Data are expressed as means±SEM. Two‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. AngII indicates angiotensin II; PRCP‐KO, prolylcarboxylpeptidase knockout; and WGA, wheat germ agglutinin.

Figure 3. Prolylcarboxylpeptidase localizes in lysosomes and mediates intracardiac angiotensin II degradation through the lysosomal pathway.

A, Immunostaining of prolylcarboxylpeptidase (red) and Lamp1 (lysosome marker) in H9C2 cells shows prolylcarboxylpeptidase partially resides in lysosomes. White arrows indicate colocalization of prolylcarboxylpeptidase and Lamp1 (yellow dots) (scale bar: 15 μm). B, Cytosolic and lysosomal fractions of wild‐type hearts show the localization of prolylcarboxylpeptidase in both cytosol and lysosome. Lamp1 is used as a marker for lysosomes. C, Angiotensin II levels in plasma. D, Angiotensin 1‐7 levels in plasma. E, Angiotensin II levels in myocardial tissues. F, Angiotensin 1‐7 levels in myocardial tissues (n=8 mice per group). Data are expressed as means±SEM. Two‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. AngII indicates angiotensin II; DAPI, 4′, 6‐diamidino‐2‐phenylindole; Lamp1, lysosomal‐associated membrane protein 1; PRCP, prolylcarboxylpeptidase; and PRCP‐KO, prolylcarboxylpeptidase knockout.

Figure 4. Prolylcarboxylpeptidase‐knockout hearts revealed more reactive oxygen species production and apoptosis than wild‐type hearts in response to 2 weeks of angiotensin II infusion.

A, Dihydroethidium staining was used to detect levels of reactive oxygen species production (scale bar: 30 μm), followed by quantification (n=8 per group). B, TUNEL staining assay was applied to detect levels of apoptotic cells in heart cross sections (scale bar: 30 μm). TUNEL: green; nuclei: blue/DAPI; a‐actinin: red. Arrows indicate TUNEL‐positive nuclei. C, The quantification of TUNEL positive nuclei is shown in bar graphs (n=7–8 per group). D, Immunoblot analysis shows expression levels of several mitogen‐activated protein kinase proteins, including eNOS, pPKB, tPKB, pERK1/2, tERK1/2, pJNK, tJNK, pMKK7, tMKK7, pMKK4, tMKK4. Student's t‐test or 2‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. AngII indicates angiotensin II; DAPI, 4′, 6‐diamidino‐2‐phenylindole; eNOS, endothelial nitric oxide synthase; ERK1/2, extracellular signal‐regulated kinases1/2; JNK, c‐Jun N‐terminal kinase; MKK4, mitogen‐activated protein kinase kinase 4; MKK7, mitogen‐activated protein kinase kinase 7; p, phhosphorylation; PKB, protein kinase B; PRCP‐KO, Prolylcarboxylpeptidase knockout; t, total protein; and TUNEL, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling.

Figure 5. AAV9‐mediated restoration of cardiac prolylcarboxylpeptidase expression alleviated the progression of angiotensin II–induced hypertrophic remodeling in the prolylcarboxylpeptidase‐knockout heart.

A, Diastolic and systolic blood pressure. B, Myocardial angiotensin II concentration. C, Wheat germ agglutinin staining of heart cross‐sections (scale bar: 50 μm). D, Echocardiographic assessment of fractional shortening %. E, Masson's trichome staining of representative myocardial sections (scale bar: 45 μm) and quantification of interstitial fibrosis. Data are presented as means±SEM. Student's t‐test or 2‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. AngII indicates angiotensin II; AAV9, adeno‐associated virus serotype 9; GFP, green fluorescent protein; PRCP, prolylcarboxylpeptidase; and PRCP‐KO, prolylcarboxylpeptidase‐knockout.

Figure 6. The combination therapy of AAV9‐mediated prolylcarboxylpeptidase overexpression and LSRT gave better efficacy than the monotherapies to treat angiotensin II–induced cardiac malfunction.

A, Diastolic and systolic blood pressure. B, Left ventricle end‐diastolic posterior wall. C, Echocardiographic assessment of fractional shortening percentage. D, Wheat germ agglutinin staining of heart cross sections. E, Masson's trichome staining for the quantification of interstitial fibrosis and terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling assay to detect levels of apoptotic cells. F, Angiotensin II levels in plasma and myocardial tissues. n=5 for angiotensin II + LSRT group and n=8 for other groups. Data are presented as means±SEM. One‐ or 2‐way ANOVA with Bonferroni correction for post hoc comparisons was used for analysis. *P value: angiotensin II + AAV9‐prolylcarboxylpeptidase vs angiotensin II + AAV9‐prolylcarboxylpeptidase + LSRT; **P value: angiotensin II + LSRT vs angiotensin II + AAV9‐prolylcarboxylpeptidase + LSRT. AAV9 indicates adeno‐associated virus serotype 9; angiotensin II, angiotensin II; GFP, green fluorescent protein; LSRT, losartan; and PRCP, prolylcarboxylpeptidase.

Figure 7. Schematic figure proposing prolylcarboxylpeptidase cardioprotection against hypertensive cardiac remodeling.

A, At a normal state, the cardiac function is regulated by angiotensin II, which is produced in the circulatory system and in cardiomyocytes. ACE2 and prolylcarboxylpeptidase work synergically to protect the hearts from the overaction of angiotensin II at both systemic and intracellular levels. B, In hypertensive diseases, the angiotensin II levels, however, are significantly upregulated due to loss of cardiac ACE2 and prolylcarboxylpeptidase expressions, leading to the development of hypertrophic remodeling and heart failure. C, In this study, we proposed that a combination treatment of AAV9‐mediated overexpression of prolylcarboxylpeptidase and losartan could ameliorate cardiac functions by improving the regulation of angiotensin II at both systemic and intracellular levels. AAV9 indicates adeno‐associated virus serotype 9; ACE, angiotensin‐converting enzyme; ACE2, angiotensin‐converting enzyme 2; AGT, angiotensinogen; AngII, angiotensin II; and ROS, reactive oxygen species.