The Effects And Mechanism Of Profilin-1in Hypertension Induced Cardiac Hypertrophy

PART ONE COMPARATIVE PROTEOMICS ANALYSIS OF CARDIAC HYPERTROPHY IN SPONTANEOUS HYPERTENSIVE RATSBackgroundMyocardial hypertrophy is a compensatory response of cardiomyocytes and extracellular matrix to the hemodynamic overload and neurohumoral disorders. It is also a common pathological process of many cardiovascular diseases, such as high blood pressure, heart valve disease, acute myocardial infarction and congenital heart disease. Clinical epidemiological data showed that myocardial hypertrophy results in significantly increased incidence of sudden cardiac death and heart failure, causing poor prognosis of patients. Myocardial hypertrophy has been listed as an independent risk factor for cardiovascular morbidity and mortality. Therefore, to explore the mechanisms of myocardial hypertrophy and find effective measures of prevention and treatment of myocardial hypertrophy is of great clinical significance and research value.Proteomics is a science with the whole proteome as research object. The aim of proteomics is to explore all the proteins in cells and their dynamic changes and to reveal the essence of life and the rule of activity in the overall level of cell and living organisms. The isobaric tags for relative and absolute quantitative techniques (iTRAQ) is a new quantitative study of comparative proteomics technology developed in recent years. iTRAQ technology can supply accurate quantitation and identification of proteins from a whole genome, search for differentially expressed proteins, and analyzes the protein function. Compared with the traditional quantitative technique dependent on gel electrophoresis, iTRAQ is of high sensitivity and accuracy based on tandem mass spectrometry method; It can do absolute and relative quantitative analysis of four and even eight different samples at the same time; Not only cytoplasm protein but also membrane protein, nucleoprotein and extracellular protein can be detected; The proteins of low abundance, strong alkaline, less than10kD or greater than200kD can all be detected by iTRAQ. The development of iTRAQ technology provides an effective and reliable method to study the mechanism of myocardial hypertrophy and look for the key proteins.Spontaneously hypertensive rats (SHR) is a genetic hypertensive animal model founded by Okamoto and Aoki of Japan’s Kyoto university in1959. SHR provide an excellent model system to investigate hypertension-mediated cardiac hypertrophy because of their similarities to human hypertension and natural development of the pathophysiological alterations also happened in patients with essential hypertension. Cardiac hypertrophy is one of the important characteristics of SHR. Hypertensive myocardial hypertrophy occurs in juvenile SHR and exists during the whole course of high blood pressure.Therefore, in this study we choosed SHR of specific ages as research object and tried to find the differentially expressed proteins in the early development of myocardial hypertrophy by using iTRAQ technology. Then we analyzed the molecular function, classification and biological processes of the differentially expressed proteins using bioinformatics methods, selected and identified the key proteins playing critical effects on the pathogenesis of hypertensive myocardial hypertrophy, and provided effective target for prevention and treatment of hypertensive myocardial hypertrophy.Objectives1. To study the characteristics of myocardial leasion in SHR and observe the pathologic and ultrastructural changes of myocardial tissue in SHR of5and17weeks old;2. To study the differentially expressed proteins between hypertrophic myocardium of SHR and normal myocardium of WKY by using iTRAQ technology, reveal the pathophysiological mechanisms of hypertensive myocardial hypertrophy on the overall level, and screen the key proteins playing critical roles;MethodsMale SHR (n=8,4weeks old), male WKY (n=8,4weeks old), male SHR (n=8,16weeks old) and male WKY (n=8,16weeks old) were kept under observation for1week (i.e.,5weeks old and17weeks old) prior to the start of experiments:1. Blood pressure measurement:Systolic blood pressure (SBP) was measured using the noninvasive blood pressure measurement system before the euthanasia of rats.2. Cardiac mass index measurement:Rats were euthanized under anesthesia and hearts were removed and weighed. Then we dissected the left ventricule and measured the left ventricular weight-to-heart weight ratio (LVW/HW Ratio). The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as another reflection of left ventricular enlargement.3. Pathological analysis:The morphological changes of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.4. Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope.5. Proteomics (iTRAQ) analysis:Protein was extracted and digested by trypsin. The peptides were labeled with iTRAQ reagents following the manufacturer’s instructions (Applied Biosystems,114for WKY-5group,115for SHR-5group,116for WKY-17group and117for SHR-17group). The labeled peptides were separated by Strong Cation Exchange chromatography and desalted by a C18column. MALDI-TOF/TOF and MicroQ-TOF mass spectrometry was used for the identification of peptides. The different ionic strength of reporter group represents the different abundance of its labeled polypeptides.6. Gene Ontology (GO) analysis of the differentially expressed proteins:Mass spectrum data was analyzed using Data Analysis software4.0and the matched proteins were searched with Mascot software. GO analysis of the differencially expressed proteins, including molecular function, biological process and cell composition, was performed using Panther software (http://www.pantherdb.org/);7. Profilin-1expression:Real-time quantitative polymerase chain reaction (qPCR) and western blot technology were used to detect the mRNA and protein level of profilin-1in each group.Results1. SBP of rats:The SBP of SHR-5group (143.1±10.5mmHg) is significantly higher than that of WKY-5group (111.2±7.6mmHg, P<0.001). The SBP of SHR-17group (216.3±12.1mmHg) is significantly higher than that of WKY-17group (117.7±8.6mmHg, P<0.001).2. Cardiac mass index measurement:The LVW/HW Ratio of WKY-5group is0.54±.02while the LVW/HW Ratio of SHR-5group is0.55±0.02.No significant differences between the two groups were found. The LVW/HW Ratio of SHR-17group (0.77±0.66) is28.3%higher than that of WKY-17group (0.60±0.03), which is significantly different. The left ventricular long axis of WKY-5group is6.42±0.36mm while the left ventricular long axis of SHR-5group is6.45±0.33mm. No significant differences between the two groups were found. The left ventricular long axis of SHR-17group (13.31±0.67) is33.5%higher than that of WKY-17group (9.97±0.60), which is significantly different.3. Pathological analysis:The myocardial cells of WKY rats and5-week-old SHR showed normal morphology. The arrangement of myocardial cells was neat and dense, with no fiber fracture. The staining of intercalated disc and nuclei are clear. SHR-17rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. Sirius red staining showed that fibrillar collagen in hearts of SHR-17group (2.93±0.26%) was much more evident than that in SHR-5group (0.15±0.01%), WKY-5group(0.13±0.01%) and WKY-17group (0.18±0.02%, P<0.001).4. Ultrastructural examination:The ultrastructure of myocardium in WKY-5and WKY-17group was normal. Although no typical hypertrophic changes were found in SHR-5rats, the cytoskeleton of myocardial cells started to show changes: the parallel and linear arrangement of the Z-discs in SHR-5rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. The nuclei and other organelles showed normal morphology. Myocytes of the SHR-17group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Rough endoplsmic reticulum and Golgi apparatus is well developed, suggesting that the synthesis and secretion is active in myocardial cells.The ultrastructral morphology of SHR-17group is consistent with that of cardiac hypertrophy. 5. Mass Spectrometry Identify the Differentially Expressed Proteins We identified506proteins in this study. Seven proteins were shown to have significantly different abundance between SHR-5group and WKY-5group, of which3were upregulated and4were downregulated. Twenty eight proteins were shown to have significantly different abundance between SHR-17group and WKY-17group, of which17were upregulated and11were downregulated. There are three mutual differentially expressed proteins:Profilin-1, Myosin light chain kinase2and Mitochondrial NADP+-isocitrate dehydrogenase.6. GO analysis of differentially expressed proteins:According to the molecular functional annotation, the differentially expressed proteins are mainly involved in structural molecule function (36.7%), binding function (30.0%) and catalytic activity (20.0%). According to the biological processes, proteins are mainly involved in cellular processes (18.0%), metabolic processes (16.0%), biogenesis (14.0%) and developmental process (14.0%). According to the cellular components,47.6%in cytoplasm,42.9%in organelles,4.8%on cell membrane and4.8%in the cellular junction. Panther protein class analysis showed that the differentially expressed proteins mainly include cytoskeletal proteins (33.3%), enzyme regulatory proteins (13.3%), structural proteins (10.0%) and calcium binding proteins (6.7%) etc. The results showed that cytoskeletal proteins play critical roles in the development of myocardial hypertrophy in SHR rats.7. Profilin-1expression levels:Real-time PCR results showed that the mRNA level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Western blot showed that the protein expression level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Abundance of profilin-1protein shows a similar trend as that iTRAQ.Conclusion1. The myocardial hypertrophy model of17-week-old SHR was successfully built, which was confirmed by cardiac mass index measurement, myocardial pathology analysis and ultrastructural observations.2. As a new technology of quantitative comparative proteomic, iTRAQ has high accuracy, repeatability and quantitative effect. It can be used to explore the molecular mechanisms of myocardial hypertrophy and look for the key proteins and pathways.3. Mass spectrometry identifed28differentially expressed proteins between 17-week-old SHR rats with hypertrophic myocardium and17-week-old WKY rats with normal myocardium. GO analysis showed that the cytoskeleton proteins in SHR rats play critical roles in the pathological process of myocardial hypertrophy.4. Real-time quantitative PCR and Western blot examination confirmed the high expression level of profilin-lin myocardial tissues of SHR rats, which verified the reliability of iTRAQ technology and provided key candidate targets for further research on the pathogenesis of myocardial hypertrophy.Part TwoThe Effects and Mechanism of Profilin-1in Hypertension Induced Cardiac HypertrophyBackgroundHigh blood pressure is the first cause of myocardial hypertrophy. When the heart is under hemodynamic overload, myocardial cells can perceive the tension and generate and release a variety of cytokines. These cytokines will combine with corresponding receptors on the cell membrane and activate a variety of hypertrophy signaling cascade. Finally the nuclear oncogene transcription is activated and myocyte hypertrophic process starts. Functional membrane proteins, cytoskeleton system as well as mutual interaction among cytokines involve in the above pathophysiological process. Cytoskeleton system is the key component to perceive the mechanical stimulation and convert mechanical stress into biochemical signals. The cytoskeleton, especially actin microfilament system, is an important point to study the mechanisem of hypertension induced cardiac hypertrophyProfilin-1is an important actin binding protein. It can modulate the dynamic assembly of actin cytoskeleton according to extracellular signals. Our previous research suggested that profilin-1plays a critical role in the early myocardial hypertrophy caused by ouabain. And the proteomics analysis mentioned in part one reveals a significant upregulation of profilin-1expression in early hypertrophic myocardium of spontaneously hypertensive rats compared with the control group. In this project, we constructed adenovirus vectors carrying profilin-1mRNA and siRNA separately, and injected them into spontaneously hypertensive rat models to induce the overexpression and silence of profilin-1gene. By this way, we studied the effects of profilin-1in the development of hypertension-induced myocardial hypertrophy and its possible mechanisms using various pathological and histologic technologies as well as molecular biology. These results will provide a new target for early prevention and treatment of hypertensive myocardial hypertrophy and is of great academic significance and potential clinical value.Objectives1. To investigate the improvement/deterioration of hypertension-induced myocardial hypertrophy by transfection of interference adeno virus vector pAd-miR-profilin-1/overexpression adeno virus vector pAd-profilin-1-IRES-EGFP into spontaneously hypertensive rats;2. To explore the mechanism by which profilin-1silencing/overexpression would ameliorate/deteriorate hypertension-induced myocardial hypertrophy on molecular, histological and ultrastructural levels.Methods1. Five-week-old boy SHR and WKY rats were randomly assigned to four groups (n=20per group):WKY-C (WKY control rats treated with negative control adenovirus); SHR-C (SHR control rats treated with negative control adenovirus); SHR-I (SHR treated with adenovirus vector pAd-miR-profilin-1to knockdown profilin-1expression); and SHR-H (SHR treated with adenovirus vector Adprofilin-1-IRES-enhanced green fluorescent protein (EGFP) to overexpress profilin-1). The adenoviruses were injected twice into tail veins of rats at a dosage of3×109infectious units per rat with an interval of6weeks between injections. Six weeks after the second administration of adenovirus, subsets of animals were randomly selected and used for cardiac mass index measurement, histopathologic and ultrastructural observation, or biochemical analysis.2. Blood pressure measurement:SBP was measured with the Heart Rate and BP Recorder for Rats, using the tail-cuff method before the start of treatment and weekly during treatment.3. Cardiac mass index measurement:Rats were weighed and euthanized under anesthesia at17weeks of age, and hearts were removed and weighed. Atria were cut away and right ventricular free walls were carefully dissected from the left. The intraventricular septum was included in the left ventricular weight. The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as a reflection of left ventricular enlargement.4. Transfection efficiency detection:Ten days after the second injection of adenovirus, five rats in each group were euthanized and their hearts removed to determine transfection efficiency. EGFP expression in the cardiac tissues was observed with a fluorescence microscope to determine transfection efficiency. For each sample, EGFP expression and transfection efficiency were evaluated in six randomly chosen fields per section and quantitated using Image-Pro Plus6.0software.5. Pathological analysis:The morphologicalchanges of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.6.Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope. Individual myocyte membranes (n=15for each group) were imaged at×40000magnifications, and the number of caveolae per micrometer plasmalemmal membrane (/μm) were counted by blinded investigators.7. Immunohistochemical and immunofluorescence staining:The expression of profilin-1in myocardium was viewed by immunohistochemical staining under a light microscope. The colocalization of caveolin-3and actin microfilament was confirmed using immunofluorescence staining.8. Measurement of nitric oxide production in serum and left ventricle:A serous and cardiac NO assay was performed using the NO assay kit. The homogenates of left ventricular samples and serum were centrifuged and the supernatant was taken for NO assay. With this method, the nitrate present in the sample was converted into nitrite by the enzyme nitrate reductase.The nitrite reacted with sulphanilamide and N-(L-naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo dye. Then the diazo dye was measured at the absorbance of550nm and the content of nitrite was obtained to reflect the production of NO. Results were expressed as μmol/g protein.9. Real-time quantitative RT-PCR:Total RNA of the rat myocardium tissues was extracted by Trizol. The mRNA expression levels of profilin-1, eNOS and caveolin-3in the left ventricle of rats were examined by real-time PCR. 10. Western blot analysis:The protien expression levels of profilin-1, eNOS, p-eNOS and caveolin-3in the left ventricle of rats were examined by western blot analysis.Results1. Effect of profilin-1on SBP of spontaneous hypertensive rats:The effect of adenovirus-mediated profilin-1gene overexpression and knockdown on SBP was monitored in SHR and WKY before injection and every week afterward. There were significant differences in SBP between SHR and WKY throughout the study (P<0.001). However, no significant differences were observed among the three SHR groups throughout the experiment.2. Cardiac mass index measurement:Compared with WKY-C group, SHR-C group showed a27%increase in left ventricular weight-to-heart weight ratio (LVW/HW Ratio) and a29%increase in the left ventricular long axis measurement. The SHR-I group displayed a significantly decreased LVW/HW Ratio when compared with the SHR-C group (P<0.05), indicating that lowering profilin-1expression reversed cardiac hypertrophy. The left ventricular long axis, another measurement of left ventricular enlargement, was also significantly reduced in SHR-I group compared with control SHR (P<0.01).3. Detection of adenovirus transfection efficiency:The high transfection efficiency was confirmed by observing EGFP expression under the fluorescence microscope. In all four groups, the green fluorescent cells accounted for a proportion of30%-35%. Meanwhile, it’s hard to see any EGFP expression in myocardial tissue of rats injected with saline.4. Pathological analysis:Compared with WKY-C rats, SHR rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. To further confirm the effects of profilin-1on cardiac hypertrophy and fibrosis, we overexpressed profilin-1in the cardiac tissue of SHR using an adenovirus carrying the profilin-1gene.The myocardium from the SHR-H group developed significant hypertrophy with cardiomyoliposis. Sirius red staining showed that fibrillar collagen in hearts of SHR-H group was much more evident than that in SHR-C group (P<0.001). These results demonstrate that high levels of profilin-1promote cardiac hypertrophy and fibrosis.5. Ultrastructural examination:Compared with WKY-C group, the parallel and linear arrangement of the Z-discs in SHR rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Myocytes of the SHR-H group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Distortion of nuclear shape was persistent and chromatin condense became more serious. The chromatin constituted a thick layer in the inner boundary of the nuclear membrane.6. Effects of profilin-1on actin cytoskeleton:Phalloidin staining in the WKY-C group was abundant but became weaker and thinner in SHR-C group. Downregulation of profilin-1expression helps to conserve the abundance of actin filaments in SHR, whereas overexpression of profilin-1further reduced actin filaments organization.7. Profilin-1overexpression decreased the caveolar abundance:Transmission electron microscopy was performed to quantify caveolar abundance. Caveolae were three-fold more abundant in WKY-C group compared with SHR-C group (P<0.001). Compared with the control SHR, transgenic overexpression of profilin-1in SHR caused a further significant reduction of sarcolemmal caveolae (P<0.001). In contrast, downregulation of profilin-1expression helps to conserve the abundance of caveolae in SHR (P<0.001). This result was consistent with the abundance of caveolin-3mRNA and protein in cardium. Both mRNA and protein expression of caveolin-3were lower in hearts of SHR compared with those in WKY. The reduction in caveolin-3expression was more severe in the SHR-H group with profilin-1overexpression. In contrast, caveolin-3expression was restored in the SHR-Ⅰ group following profilin-1knockdown.8. Profilin-1expression levels and cellular localization in the myocardial tissue of rats:Real-time PCR results showed that the mRNA level of profilin-1in SHR-H group is significantly higher than that in SHR-C group (P<0.05) while the mRNA level of profilin-1in SHR-Ⅰ group is significantly lower than that in SHR-C group (P<0.05). Western blot showed that the protein expression level of profilin-1in four groups is similar as that of mRNA. The immunohistochemical analysis of profilin-1in the heart of SHR and WKY showed that profilin-1protein was expressed in the cytoplasm of cardiomyocytes, especially around the nucleus. Abundance of profilin-1protein shows a similar trend as that in western blot analysis. We also observed mild profilin-1expression in vascular smooth cells.9. Content of NO in serum and myocardial tissue:NO secretion was lower in hearts of SHR compared with WKY. We investigated production of NO in left ventricular and found a significant decrease in SHR-H group (P<0.01) and a significant increase in SHR-I group (P<0.05) when compared with SHR-C group. We also tested the NO levels in the serum of rats and no significant differences were found among the SHR-C group, SHR-I group, and SHR-H group, indicating that the changes of NO levels is a local phenomenon rather than a systemic reaction.10. Expression and activity of eNOS in rat myocardial tissue:No significant difference of eNOS expression was found among the four treatment groups. eNOS activity and NO secretion were lower in hearts of SHR compared with WKY. Our results showed a significant decrease of phospho-S1177eNOS in SHR-H group and a significant elevation in SHR-I group, when compared with SHR-C group (P<0.05).Conclusion1. Profilin-1expression is upregulated in hypertrophic myocardium of spontaneously hypertensive rats, accompanying with cardiomyocyte hypertrophy, hyperplasia of collagen fibers and actin cytoskeleton damage.2. Overexpression of profilin-1interfered with the assembly of actin cytoskeleton and the formation of caveolae on myocardial membrane, then inhibited the activity of the eNOS/NO signaling pathways and finally promoted hypertension-induced myocardial hypertrophy. This might be one of the most important pathogenesis of myocardial hypertrophy.3. Profilin-1gene silencing could effectively reverse myocardial hypertrophy by increasing the content of myocardial actin microfilament, increasing the caveolar number and restoring eNOS activity and the product of NO.