Digital Theses Archive


Tesi etd-03292019-114235

Type of thesis
Cardiac remodeling in models of anthracycline-induced cardiomyopathy
Scientific disciplinary sector
SCIENZE MEDICHE - Translational Medicine
  • anthracyclines
  • cardiac magnetic resonance
  • cardiomyopathy
  • cardiooncology
  • preclinical
Exam session start date
Abstract<br>Background: <br>Anthracyclines (AC) represent the cornerstone of treatment of many different malignancies. Unfortunately, and despite their extreme usefulness as antineoplastic agents, their use is being hampered by their collateral effects with AC induced cardiomyopathy being one of the most important. Many AC treated oncologic patients, are at increased risk for developing sooner or later a cardiac related disease. Given this premises, it is clear why AC induced cardiomyopathy has been and still is one of cardiology’s most active research fields. Scientific efforts are constant, trying to improve our ability to treat this disease, to detect it as early as possible and to prevent it from occurring. Animal models therefore are of paramount importance and necessary for further understanding all possible c pathways involved, developing diagnostic techniques and testing potential cardioprotective treatments.<br>Aim of the study:<br>To develop a small animal model of AC induced cardiomyopathy, to test different in-vivo and ex-vivo diagnostic techniques for cardiac involvement assessment and to compare the potential efficacy of three different possible cardioprotectors, enalapril, ranolazine and spironolactone. To develop the first large animal model of chronic AC induced cardiomyopathy and to test different in-vivo and ex-vivo diagnostic techniques for cardiac involvement assessment.<br>Materials and methods:<br>A murine model of AC-induced cardiomyopathy has been developed, and studied by a 2-step protocol consisting in: a) a dose-response protocol aiming to identify the best AC treatment regimen. For this reason, animals were treated with 2 different AC doses over 10 weeks period, undergoing echocardiographic assessment every 2 weeks. Two animals were randomly sacrificed every 2 weeks and blood sample collected for cardiac biomarkers analysis (high-sensitive cardiac troponin T (HS-TnT) and galectin 3). Heart was explanted for ex-vivo analysis for mitochondrial functional (calcium homeostasis assessed by ryanodine receptors concentration and their affinity to ryanodine) and structural (transmission electron microscope (TEM)) assessment; b) the best AC treatment regimen was then used for testing and comparing 3 different drugs (enalapril, ranolazine and spironolactone) administered for 5 weeks on top of AC treatment in animals already AC pre-treated for 5 weeks.<br>Furthermore, a large animal model was developed with pig representing the selected species. Different doses of AC were tested in order to develop a porcine animal model of AC cardiomyopathy. The best AC treatment regimen was then used for describing the presence and extent of cardiac involvement by means of in-vivo cardiac magnetic resonance (CMR) assessment, performed at baseline and at treatment end, and ex-vivo assessment, as previously described for the murine animal model.<br>Results:<br>In the murine model, 20 mice were divided in 2 groups, one group treated with 2 and one with 3 mg/kg/week of doxorubicin for 10 weeks. Overall mortality was low (5%) and animals tolerated well both doses. Serial echocardiographic scans demonstrated a reduction in left ventricular ejection fraction (LVEF) that was statistically significant in both groups between baseline and end of treatment (p&lt;0.05) without significant difference between groups. Ex-vivo characterization documented similar results also regarding HS-TnT and TEM-documented ultrastructural changes. <br>In the second part of the small animal study, dose of 2 mg/kg/week was used for 5 weeks in 32 animals. Subsequently, 4 groups (8 animals each) were formed and continued treatment for another 5 weeks. AC group received AC, ENA group received enalapril and AC, RLZ group received ranolazine and AC and SPI group received spironolactone and AC. Moreover, a control group treated with only saline throughout the study was formed. Mortality was slightly higher but similar in all groups (1 death per group except from SPI group with 2 deaths). Echocardiography showed a significant reduction in LVEF in all animals treated with AC until the fifth week with delta LVEF (difference of LVEF between LVEF at 5 weeks and at treatment end) acting differently among groups. LVEF remained stable in the control group (Delta LVEF -1.86%  1.3). LVEF continued to decrease in AC group (Delta LVEF -12.9%  2.9), whereas in RLZ showed a slight decrease (Delta LVEF -4.7%  1.6). In SPI group LVEF plateau (Delta LVEF -0.1%  5.4) whereas in ENA group LVEF showed a slight recovery (Delta LVEF 4.4%  1.9). The only significant differences were those between control and AC groups and between ENA and AC groups (both p&lt;0.05). In ex-vivo analysis, significantly lower values of HS-TnT in the treated groups were observed with each one of the cardioprotective drugs (with no statistical difference among groups) when compared with AC group. Calcium homeostasis assessment showed no significant differences in terms of ryanodine receptors number and their affinity among various groups. On TEM analysis, treatment with RLZ or SPI was able to induce a medium and low degree of recovery/protection respectively. ENA treated animals, showed ultrastructure very similar to that of the control group with diffuse evidence of autophagic vacuoles observed inside the cardiomyocytes, generally containing mitochondria (mitophagy) and a high degree of recovery/protection from AC induced cardiomyopathy. This qualitative evidence was also quantitively confirmed with a significant increase in the volume density of the mitochondria in the cardiomyocytes of AC group. Main alteration of mitochondria was volume increase only partially corrected in RLZ group and fully recovered in ENA group. Number of autophagy vacuoles was significantly increased in ENA group (p&lt;0.05).<br>Regarding the large animal model, in the first part of the study, different groups of animals treated with different AC doses and dose regimens were considered. Animal treated with 1.2 mg/kg/week over 6 weeks showed no cardiac damage whereas animals treated with 2 mg/kg/week over 6 weeks died prematurely for extracardiac causes. Animals treated with 2 mg/kg every 2 weeks for 8 weeks showed high and unexpected mortality without evidence of reproducible cardiac involvement. The group treated with 1.6 mg/kg/week over 6 weeks was selected as the most appropriate. A total of 8 pigs were analysed with a high mortality (37,5%). All animals underwent CMR before and all animals completing observation period (5 animals) at the end of AC treatment. LV dimensions and systolic function remained stable and normal throughout treatment in all animals and none developed myocardial edema or fibrosis. Three out of 5 animals developed significant diastolic dysfunction (2 restrictive pattern and 1 pseudonormal filling pattern). HS-TnT showed a slight and non-significant increase up to 7.6  5.9 ng/L before sacrifice whereas galectin-3 baseline values of 1.2  0.6 ng/mL remained stable at 1.3  0.8 ng/mL at treatment end. Calcium homeostasis assessment revealed a significant difference in terms of total number of ryanodine receptors as assessed by means of Bmax (103  34 fmol/mg in the control group vs 41  10 fmol/mg in the AC group (p&lt;0.05)). Ryanodine affinity assessed by means of Kd, did not differ significantly between control (8.1  12.5 nM) and AC (1.6  1.9 nM) group. <br>Conclusions:<br>Murine animal model is a valid AC induced cardiomyopathy small animal model. AC induced cardiomyopathy can be documented by different in-vivo and ex-vivo techniques. Multiple pathophysiological pathways coexist and intersect in AC cardiomyopathy with mitochondrion being the leading actor of all mechanisms involved. Among tested medications, enalapril has proven to provide a significant cardioprotective aid, being able to impede LVEF reduction when compared with AC-treated animals. On TEM, an increase both in number and volume density of autophagic vacuoles (mitophagy), in ENA group could explain a potential reparative mechanism by means of the removal of damaged mitochondria altered by AC treatment. Porcine animal model is a suboptimal large animal model for AC cardiomyopathy given the low reproducibility of cardiac involvement and the high mortality of AC treated animals. In the best porcine AC cardiomyopathy model developed, a troponin increase and altered calcium homeostasis was observed. Moreover, systolic function remained within normal limits in all animals, but significant diastolic dysfunction occurred in the majority of AC-treated animals.<br>