Cardiac Protective Effects Of Thermosensitive Hydrogel/Angiotensin-Converting Enzyme Inhibitors Post Myocardial Infarction In Rats

Poly N-isopropylacrylamide hydrogel (PNIPAAm hydrogel) is a temperature-sensitive hydrogel, synthesized by isopropylacrylamide muti-chain, caprolactone mutil-chain, hydroxyethl methacrylate and dextran.PNIPAAm hydrogel achieves the structural support due to its similar three-dimensional structure as native extracellular matrix of normal heart. Besides that, it can improve the local microenvironment through promote neovascularization formation in the infarcted myocardium, reduce the pressure of ventricular cavity due to thickened the ventricular wall of infarct zone, regulate the collagen metabolism due to the exogenous collagen complication, enhance the contractility of isolated infarcted myocardium through the improved systolic and relaxation coordination, potentially reduce the incidence of malignant ventricular arrhythmias due to reducing the electrical instability and inhibiting the electrical heterogeneity of infarcted myocytes, finally, achieve the cardiac protection.Angiotensin-converting enzyme inhibitor (ACEI) is a classic therapeutic strategy for coronary heart disease patients. Although the different pharmacokinetic characteristics of ACEI showed the theoretical and potential difference of its functional power and lasting time, the pharmacological advantage whether realize the clinical endpoints improvement has yet to be confirmed and need to further researched.Part1Physical Features of Thermosensitive Poly N-isopropylacrylamide Hydrogel Promote its Effects on Cardiac Protection post Myocardial InfarctionObjectivesThe negetive ventricualr remodeling post myocardial infarction due to over degradation of extrocellular matrix and the formation myocardial fibrosis is one of the most crucial factors which can affect the prognosis of patients suffered from myocardial infarction. Previous researches have exhibited the protective effects of biomaterials for myocardial infarction (MI). However, the relevance between the inherent features of biomaterials and their function is still not completely understood. In this study, we aim to investigate the interation of physical charateristics and function of Poly N-isopropylacrylamide Hydrogel (PNIPAAm Gel).MethodsTwo types of PNIPAAm Gel with different degradation times (defined as Gel A and Gel B) were synthesized. In vivo hydrogel formation and maintenance were observed and confirmed. Left anterior descend of coroanry artery in rats was ligated to make myocardial infarction models in rats. Then, all the mycardial infarction induced rats were divided into MI+Phosphate Buffer Solution group, MI+GelA group and MI+GelB gorup randomly. During the first five minutes after infarcion,100μl Phosphate Buffer Solution (PBS, pH7.4),100μl3%(w/w) GelA solution,100μl3%(w/w) GelB solution was injected into the infarcted myocardium as desigend, respectively. Rats in sham operation group received the same process, except for the left anterior descend coronary atrety was not ligated and received100μl3%(w/w) GelA solution,(Sham+GelA group)100μl3%(w/w) GelB solution (Sham+GelB group), respectively. All rats were fed for ninety days and then the rats received ultrasonic cardiogram, haemodynamics, isolated myocardium contraction ability, H&E and immunohistochemistry staining test.ResultsPNIPAAm Gel exhibited similar geometrical construction as native extracellular matrix (ECM). Both GelA and GelB enhanced the contractility of isolated infarcted myocardium [MI+GelA group (80.09±1.19) mg, MI+GelB group (80.81±4.96) mg vs. MI+PBS group (40.27±0.8)mg, P<0.05for all comparisons], shorten the increased time of isolated myocardium under the condition of electrical stimulation [mean systolic time:MI+GelA group (107.77±1.83) ms, MI+GelB group (105.15±2.16)ms vs. MI+PBS group (122.92±1.50)ms; mean systolic-diastolic whole time:MI+GelA group (303.72±1.99)mg, MI+GelB group (300.74±1.99)mg vs. MI+PBS group (341.36±3.51)ms, P<0.05for all comparisons], increased neovascularization [anti-a-SMA mean IOD:MI+GelA group (0.0394±0.0020), MI+GelB group (0.0393±0.0070) vs. MI+PBS group (0.0152±0.0023), P<0.05for all comparisons], and finally inhibited left ventricle expansion and improved cardiac function [LVEDD:MI+GelA group (77.13±2.45) mm, MI+GelB group (69.88±3.59)mm vs. MI+PBS group (107.57±4.85) mm; LVESD MI+GelA group (51.0±5.37)mm, MI+GelB group (43.71±4.53)mm vs. MI+PBS group (86.86±3.7)mm; The thickness of infarcted myocardium: MI+GelA group (15.0±2.02)mm, MI+GelB group (16.25±1.06)mm vs. MI+PBS group (7.57±0.69)mm; left shortening fraction:MI+GelA group (31.37±5.86)%, MI+GelB group (32.95±2.33)%vs. MI+PBS group (18.51±3.50)%; the minimum and maximum rate pressure change in the left ventricular:Dp/Dt MI+GelA group (3107.46±131.53)mmHg/s, MI+GelB group (3925.05±73.74)mmHg/s vs. MI+PBS group (2596.94±253.85)mmHg/s;-Dp/Dt MI+GelA group (3026.20±158.74)mmHg/s, MI+GelB group (3848.27±69.70)mmHg/s vs. MI+PBS group (2495.78±234.80)mmHg/s, P<0.05for all comparisons]. MI+GelB showed better performance on enhancing myocardial contractility and reducing the minimum and maximum rate pressure change in the left ventricular.ConclusionsIntra-myocardial injection of PNIPAAm Gel achieves the structural support due to its similar three-dimensional structure as native extracellular matrix of normal heart and some functional repair of damaged extracellular matrix. PNIPAAm Gel is a proper biomaterial for heart tissue engineering due to its good biocompability, suitable degradation rate and designed physical properties. Its physical properties promote its cardiac protection function. Part2the Effect of Thermosensitive Poly N-isopropylacrylamide Hydrogel on Collagen Metabolism post Myocardial InfarctionObjectivesExtracellular matrix is the most important part to maintain the normal structure of heart, promote the growth, differentiation and function of myocardial myocytes. Furthermore, collagen is the crucial component of extracellular matrix. In this study, we aim to assess the influence of PNIPAAm Gel on collagen metabolism.MethodsAll the experimental groups and operation process was as same as part one. After feeding for ninety days, all rats were euthanatized, and then heart tissue western blot were used to test the amount of TGF-β1protein and Real-Time RCR were used to test the explanation of collagen I and collagen III mRNA (normalized to β-actin) expressed as the collagen I/collagen III. Besides that, heart tissue was fixed and received Masson staining to show the collagen deposition in the post-infarcted myocardium.ResultsPNIPAAm Gel can significantly inhibited the deposition of collagen, expressing lower collagen volume fraction in the PNIPAAm Gel injected infarcted myocardium, compared with PBS injected infarcted myocardium [MI+GelA group (53.85+2.33)%, MI+GelB group (49.98±2.47)%vs. MI+PBS group (73.23±1.77)%, P<0.05for all comparisons]. Western blot test showed the significantly increased of TGF-β1in infarcted myocardium post MI, but whatever PNIPAAm Gel or PBS solution injection into the infarcted myocardium, the TGF-β1expression was not altered. PCR test showed the intra-myocardial injection of PNIPAAm Gel can significantly inhibited the increased the ration of collagen I/collagen III.ConclusionsThe inhibition effects of PNIPAAm Gel on collagen metabolism are not realized through the TGF-β1pathway, but we infer that is probable due to the exogenous collagen compaction effect providing by the intra-myocardium use of PNIPAAm Gel. Part3Thermosensitive Poly N-isopropylacrylamide Hydrogel Inhibits the Increased Electrical Heterogeneity of Infarcted Myocardium after Myocardial InfarctionObjectiveMalignant ventricular arrhythmia is the vital factor to assess the prognosis of patients who suffer from myocardial infarction, and the exact mechanism underlying that is unknown. The electrical instability and the heterogeneity of myocardial myocytes are considered to be associated with the incidence of malignant ventricular arrhythmias. Recent studies have demonstrated the protective effects of PNIPAAm Gel against myocardial infarction (MI) in animals, which occurs via the inhibition of ventricular structural remodeling, and also the conductivity of the PNIPAAm Gel is also demonstrated. However, it is not clear whether the PNIPAAm Gel may influence the electrical activity of infarcted or normal myocardium. The present study was performed to investigate the changes in cardiomyocytes electrophysiology that occurred when PNIPAAm Gel was used.MethodsThe PNIPAAm Gel used in this part is the Gel B synthized in part one. Rabbits were randomly divided into the MI group or sham operation (Sham) group, and they were given PNIPAAm Gel or PBS by intra-myocardial injection into infarcted or sham myocardium, respectively. The effective refractory period (ERP), monophasic action potential duration at90%repolarization (MAPD90) and transmural dispersion of repolarization (TDR) were measured at different time point (30minutes,3hours and6hours) after surgery in endomyocardium (Endo), midmyocardium (Mid) and epimyocardium (Epi). Arrhythmias were recorded by surface electrocardiogram during the first30minutes after surgery.ResultsERP and MAPD90in Endo, Mid and Epi were significantly reduced post-MI, whereas TDR was substantially increased. However, the intra-myocardial injection of PNIPAAm Gel significantly increased ERP and MAPD90in the three layers of infarcted myocardium and reduced TDR at the designated time post-MI. Consequently, the occurrence of malignant ventricular arrhythmias was reduced. Encouragingly, the PNIPAAm Gel showed no obvious influence on the electrical activity of sham-operated myocardium.ConclusionIntra-myocardium use of PNIPAAm Gel significantly inhibited the enhanced electrophysiological heterogeneity of infarcted myocardium, and such treatment is potentially useful for reducing malignant ventricular arrhythmias. Part4the Relevance between Histocompatibility of Angiotensin Converting Enzyme Inhibitor and Its Function on Inhibitory to Negative Ventricular Remodeling post Myocardial InfarctionObjectivesRenin-angiontensin-aldosterone system (RAAS) is widely evidenced its key point on regulating blood pressure and water-electrolyte metabolis. Angiontensin is an important active product of the RAAS, and the increasing expression of angioten and angiotensin-converting enzyme in the infarcted and non-infarcted zone of left ventricular myocardium are proved by previous research. The effectiveness of angiotensin-converting enzyme inhibitor (ACEI) on inhibiting the upregulated angionten II and the progress of negative ventricular remodeling have been evidenced. According to the pharmacokinetic characteristics of ACEI, benazepril (Ben), containing the solid carboxyl in combination with ACE, has the better function and more lasting ACE inhibition efficacy, compared with captopril (Cap) which contains mercapto containing binding sites. However, its clinical eligible benefits remain to be further confirmed. In this part, PNIPAAm Gel was used as a carrier, two kind of ACEI with different affinity were directly applied into the infarcted myocardium to explore the theoretical ACEI tissue affinity can truly achieve clinical benefit. MethodsThe PNIPAAm Gel used in this part is the GelB synthized in part one. Male Wiatar rats were used to make myocardial infarction (MI) animal models. The operation process was as same as part one. All MI rats were divided into MI+PBS group, MI+Gel group, MI+Gel+Cap group and MI+Gel+Ben group randomly. During the first five minutes after infarcion,100μl Phosphate Buffer Solution (PBS, pH7.4),100μl3%(w/w) Gel solution,100μl3%(w/w) Gel with Cap (50mg/ml) solution and100μl3%(w/w) Gel with Ben (10mg/ml) solution was injected into the infarcted myocardium as desigend, respectively. Rats in sham operation group received the same process, except for the left anterior descend coronary atrety was not ligated and received100μl3%(w/w) Gel solution (Sham+Gel group). All rats were fed for ninety days and then the rats received ultrasonic cardiogram, haemodynamics, isolated myocardium contraction ability, H&E, Masson and immunohistochemistry staining test. Heart tissue was used to test the expression of angiotensin-II (Ang-II) and angiotensin-converting enzyme (ACE) by Elisa.ResultsOn the aspect of inhiting left ventriclar expansion, Gel/ACEI solution perforamce better than Gel solution alone, however, there was on significant differece between Ben/Gel treated and Cap/Gel treated rat’s hearts [LVEDD:MI+Gel+Cap group (69.75±0.95) mm, MI+Gel+Ben group (70.83±3.59)mm, MI+Gel group (77.13±2.45)mm vs. MI+PBS group (107.57±4.85)mm, P<0.05; LVESD:MI+Gel+Cap group (47.5±2.4)mm, MI+Gel+Ben group (40.60±6.71)mm, MI+Gel group (51.50±5.37)mm vs. MI+PBS group (86.86±3.7) mm, P<0.05]. Although both Gel/ACEI therapy and Gel solution injection alone can significant increased the thickneness of infarcted myocardium and improved left shortening farction, there was either no significant between Gel/ACEI and Gel alone injection treatment [LV thickness:MI+Gel+Cap group (12.75±0.48)mm, MI+Gel+Ben group (13.67+2.17)mm, MI+Gel group (15.00±2.02)mm vs. MI+PBS group (7.57±0.69)mm, P<0.05; FS:MI+Gel+Cap group (31.91±3.24)%, MI+Gel+Ben group (31.49+5.74)%, MI+Gel group (31.37+5.86)%vs. MI+PBS group (18.51±3.50)%, P<0.05]. Gel/ACEI groups significantly increased the minimum and maximum rate pressure change in the left ventricular post MI, compared with Gel alone group, besides that, Cap/Gel group performance better [Dp/Dt: MI+Gel+Cap group (6195.81+68.28) mmHg/s, MI+Gel+Ben group (5418.46+64.76) mmHg/s, MI+Gel group (3925.05±73.74) mmHg/s vs. MI+PBS group (2596.94±253.85) mmHg/s, P<0.05;-Dp/Dt; MI+Gel+Cap group (5978.52±98.44) mmHg/s, MI+Gel+Ben group (4734.45±67.96) mmHg/s, MI+Gel group (3848.27±69.70) mmHg/s vs. MI+PBS group (2495.78+234.80) mmHg/s, P<0.05].Gel/ACEI groups can significantly increased the contraction force of isolated infarcted myocardium, but without any significantly differnce when compared Cap/Gel group and Ben/Gel group [MI+Gel+Cap group (107.14±1.67) mg, MI+Gel+Ben group (106.8±6.98) mg, MI+Gel group (80.81±4.96) mg vs. MI+PBS group (40.27±0.85) mg, P<0.05]. The expression of Ang-II and ACE was significantly increased post MI, Gel/ACEI groups can reduce the value significantly and the performance of Gel/Ben group was the best one among the three groups [Ang-II:MI+Gel+Cap group (803.89±89.47) ng/ml, MI+Gel+Ben group (5418.46±56.32) ng/ml, MI+Gel group (1137.15±119.33) ng/ml vs. MI+PBS group (1304.17±119.87) ng/ml, P<0.05; ACE:MI+Gel group (102.68±4.04) ng/ml, MI+Gel+Cap group (91.03±5.7) ng/ml, MI+Gel+Ben group (83.31±0.55) ng/ml vs. MI+PBS group (115.66±5.51) ng/ml, P<0.05]. While on the inhibition of infarct size, Gel/Cap group perforence the best [MI+Gel+Cap group (31.49±1.17)%, MI+Gel+Ben group (35.421.29)%, MI+Gel group (47.02±0.78)%vs. MI+PBS group (63.60+1.28)%, P<0.05]. At the aspect of neovascularization formation, there was no significant difference among the Gel/ACEI gruops and Gel alone goups [MI+GelB+Cap group (0.0409±0.0023), MI+Gel+Ben group (0.0374±0.0015), MI+Gel group (0.0393±0.0070) vs. MI+PBS group (0.0152±0.0023), P<0.05]. Also, Gel/Cap treatment can signicantly reduced the increased collagen volume farction [MI+Gel+Cap group (30.10+2.27)%, MI+Gel+Ben group (38.39±3.27)%, MI+Gel group (49.98±2.47)%vs. MI+PBS group (73.23±1.77)%,P<0.05].ConclusionsThe cinical benefits of ACEI achieved by its theoretical ACEI tissue affinity should be suspected and further discussed.