Tesi etd-01222024-112904
Link copiato negli appunti
Tipo di tesi
Dottorato
Autore
L'ABBATE, SERENA
URN
etd-01222024-112904
Titolo
Trastuzumab-induced cardiotoxicity in diverse mouse models: role of pre-existing cardiac dysfunction and efficacy of a combined beta-blocker and ACE inhibitor therapy
Settore scientifico disciplinare
BIO/09
Corso di studi
Istituto di Scienze della Vita - PHD IN MEDICINA TRASLAZIONALE
Commissione
relatore Prof. EMDIN, MICHELE
Parole chiave
- cardiac toxicity
- cardio-oncology
- cardioprotective therapy
- mouse
- trastuzumab
Data inizio appello
25/06/2024;
Disponibilità
parziale
Riassunto analitico
Background: There is incomplete knowledge about the effects of trastuzumab (TRZ) on the heart. This poses challenges for the prevention of TRZ-related cardiotoxicity, especially considering the growing prevalence of both cancer and cardiovascular disease among the ageing population. Therefore, there is a considerable interest in cardio-oncology in defining the link among cardiotoxicity, pre-existing comorbidities, and effects of cardioprotective therapy. To address this issue, experimental models are needed replicating the complexity of human cardiotoxicity and its relationship with comorbidities. However, modelling TRZ-induced cardiotoxicity in mice with pre-existing cardiac dysfunction has not been attempted before.
Aim: This project explored the relationship between pre-existing cardiac dysfunction and TRZ-induced cardiotoxicity in mouse models from low risk to overt cardiac dysfunction. Additionally, we examined whether administering an ACE inhibitor (captopril) and a beta-blocker (bisoprolol) simultaneously provides successful cardioprotection in mice models with TRZ cardiotoxicity.
Methods: Our study used 130 mice. To replicate an increased susceptibility to cardiac dysfunction in vivo, we used transgenic mice with cardiac-specific overexpression of the aldosterone synthase gene (AS mice, n=81). In a subset of AS mice, we administered isoproterenol (ISO) to induce overt cardiac dysfunction (AS+ISO, n=48). The control group consisted of WT mice (WT, n=49). Mice in each cohort were treated with TRZ (WT+TRZ, n=18; AS+TRZ, n=12; AS+ISO+TRZ, n=14) or vehicle weekly for four weeks (WT+PBO, n=14; AS+PBO n=10; AS+ISO+PBO n=14) to assess functional, structural, and ultrastructural effects of TRZ treatment. Captopril and bisoprolol (ACEi_BB) were administered in combination with TRZ treatment to evaluate the effectiveness of a cardioprotective therapy against TRZ cardiotoxicity (WT+TRZ+ACEi_BB n=6, and AS+ISO+TRZ+ACEi_BB, n=9). Cardiac electrical activity, function, and morphology were evaluated using electrocardiography, high-frequency ultrasound, histology, and electron microscopy at three different time points: baseline (T0), 10 days (T1), and 28 days (T2) after the first TRZ injection. Heart samples were collected at each time point for ex vivo examination.
Results: At T0, the AS and AS+ISO groups were compared with the control group to define their cardiac phenotype. AS mice showed increased left ventricular (LV) mass (121±17 mg vs 103±11, p<0,05 vs WT) and increased cardiomyocyte cross-sectional area (248 ± 19 μm2 vs 216±5 μm2, p< 0.05). AS+ISO mice showed a significant decrease in left ventricular (LV) ejection fraction (46±5% vs 62±3%, p<0.001 vs WT), increased LV mass (124±15 mg vs 103±11 mg, p<0.05 vs WT), and increased cardiomyocyte cross-sectional area (CSA) (271 ±19 μm2 vs 216±5 μm2, p<0.001 vs WT). Histopathological evidence of focal tissue damage and ultrastructural evidence of mitochondrial dilation, expressed by the values of mitochondria volume density (29±2% vs 21±4%, p<0.05, vs WT), were consistently observed in the AS+ISO group. These findings allowed us to define three different mouse models with levels of cardiac impairment ranging from healthy to overt cardiac dysfunction.
TRZ had a negative impact on cardiac function, structure, and ultrastructure of all three groups. At T2, there was a significant decrease in LV ejection fraction (EF) in all groups that received TRZ compared to the corresponding PBO groups. The decline was observed to be 56 ±2% vs 63 ±2% in WT, 51±1% vs 58±3% in AS, and 46±2% vs 50±2% in AS+ISO. This decline was statistically significant in all cases (p<0.001). In all groups, TRZ caused a significant increase of cardiomyocyte CSA compared to the corresponding PBO groups (265±18 μm2 in WT+TRZ; 263±11 μm2 in AS+TRZ, 308±15 μm2 in AS+ISO+TRZ; p<0.001, p<0.05, p<0.05, p<0.001, respectively). The higher values of CSA were observed in the AS+ISO group (p<0.001 for both). TRZ induced infiltration of inflammatory cells in the myocardial tissue in all groups and worsened the tissue damage and perivascular fibrosis in the AS+ISO group. Ultrastructural changes were observed in all TRZ-treated groups, including increased mitochondrial volume density (33±3%, 33±2%, 37±2% in WT, AS, and AS+ISO respectively; p<0.001 vs PBO in all the groups), and percentage of swollen mitochondria (21±3%; 21±3%; 27±4% in AS+ISO mice; p<0.001 in all cases) with higher value observed in AS+ISO+TRZ-group compared to the other TRZ-treated groups (p<0.05 for both). ACEi_BB therapy prevented the TRZ-induced decrease in LVEF in both groups (p<0.0001 vs the corresponding TRZ-treated group). In AS+ISO mice, the systolic function was significantly improved, with LVEF values reaching levels beyond those seen in the AS+ISO+PBO group (55±3% vs 50±2%, p<0.001). Both groups showed a restoration of the intervals of ventricular electrical activity, with a decrease in the duration of both QTc and JTc intervals compared to the TRZ-treated groups (p<0.0001). Additionally, an improvement in cardiomyocyte histopathological pattern and CSA was observed in both WT+TRZ+ACEi_BB (240±9 μm2 vs 265±18 μm2, p<0.05 vs WT+TRZ group) and AS+ISO+TRZ+ACEi_BB (250±7 μm2 vs 308±15 μm2, p<0.0001 vs AS+ISO+TRZ). In the latter group, cardioprotective therapy further reduced CSA to values even lower than those seen in AS+ISO+PBO (250±7 μm2 vs 284±16 μm2, p<0.01). Cardiac ultrastructure was preserved in both cases, with reduced mitochondria volume density and swollen mitochondria (p<0.0001 vs TRZ-treated group, for both). In AS+ISO+ mice, signs of activation of the lysosomal autophagic compartment were observed, with evidence of vacuoles containing damaged and dilated mitochondria.
Conclusions: Modelling cardiotoxicity in mice with pre-existing cardiac dysfunction is a valuable approach to investigate the cardiotoxic effects of TRZ and explore its relationship with pre-existing cardiac dysfunction. This approach revealed that TRZ therapy leads to systolic impairment, cardiomyocyte hypertrophy, and mitochondrial abnormalities. TRZ induced cardiac dysfunction and remodelling to a comparable extent across the groups. When TRZ cardiotoxicity acted on top of conditions of pre-existing cardiac dysfunction and/or tissue alteration, a worse cardiac phenotype was observed. Overall, these data provide an improved understanding of the extent of cellular and ultrastructural damage that result from TRZ treatment in mice. Our findings showed that captopril and bisoprolol protected against TRZ-induced cardiac remodelling and dysfunction. Notably, the beneficial the effect of captopril and bisoprolol was significant in mice with pre-existing cardiac impairment, providing valuable insights for future research on cardioprotection against TRZ-induced cardiotoxicity in the high-risk setting.
Aim: This project explored the relationship between pre-existing cardiac dysfunction and TRZ-induced cardiotoxicity in mouse models from low risk to overt cardiac dysfunction. Additionally, we examined whether administering an ACE inhibitor (captopril) and a beta-blocker (bisoprolol) simultaneously provides successful cardioprotection in mice models with TRZ cardiotoxicity.
Methods: Our study used 130 mice. To replicate an increased susceptibility to cardiac dysfunction in vivo, we used transgenic mice with cardiac-specific overexpression of the aldosterone synthase gene (AS mice, n=81). In a subset of AS mice, we administered isoproterenol (ISO) to induce overt cardiac dysfunction (AS+ISO, n=48). The control group consisted of WT mice (WT, n=49). Mice in each cohort were treated with TRZ (WT+TRZ, n=18; AS+TRZ, n=12; AS+ISO+TRZ, n=14) or vehicle weekly for four weeks (WT+PBO, n=14; AS+PBO n=10; AS+ISO+PBO n=14) to assess functional, structural, and ultrastructural effects of TRZ treatment. Captopril and bisoprolol (ACEi_BB) were administered in combination with TRZ treatment to evaluate the effectiveness of a cardioprotective therapy against TRZ cardiotoxicity (WT+TRZ+ACEi_BB n=6, and AS+ISO+TRZ+ACEi_BB, n=9). Cardiac electrical activity, function, and morphology were evaluated using electrocardiography, high-frequency ultrasound, histology, and electron microscopy at three different time points: baseline (T0), 10 days (T1), and 28 days (T2) after the first TRZ injection. Heart samples were collected at each time point for ex vivo examination.
Results: At T0, the AS and AS+ISO groups were compared with the control group to define their cardiac phenotype. AS mice showed increased left ventricular (LV) mass (121±17 mg vs 103±11, p<0,05 vs WT) and increased cardiomyocyte cross-sectional area (248 ± 19 μm2 vs 216±5 μm2, p< 0.05). AS+ISO mice showed a significant decrease in left ventricular (LV) ejection fraction (46±5% vs 62±3%, p<0.001 vs WT), increased LV mass (124±15 mg vs 103±11 mg, p<0.05 vs WT), and increased cardiomyocyte cross-sectional area (CSA) (271 ±19 μm2 vs 216±5 μm2, p<0.001 vs WT). Histopathological evidence of focal tissue damage and ultrastructural evidence of mitochondrial dilation, expressed by the values of mitochondria volume density (29±2% vs 21±4%, p<0.05, vs WT), were consistently observed in the AS+ISO group. These findings allowed us to define three different mouse models with levels of cardiac impairment ranging from healthy to overt cardiac dysfunction.
TRZ had a negative impact on cardiac function, structure, and ultrastructure of all three groups. At T2, there was a significant decrease in LV ejection fraction (EF) in all groups that received TRZ compared to the corresponding PBO groups. The decline was observed to be 56 ±2% vs 63 ±2% in WT, 51±1% vs 58±3% in AS, and 46±2% vs 50±2% in AS+ISO. This decline was statistically significant in all cases (p<0.001). In all groups, TRZ caused a significant increase of cardiomyocyte CSA compared to the corresponding PBO groups (265±18 μm2 in WT+TRZ; 263±11 μm2 in AS+TRZ, 308±15 μm2 in AS+ISO+TRZ; p<0.001, p<0.05, p<0.05, p<0.001, respectively). The higher values of CSA were observed in the AS+ISO group (p<0.001 for both). TRZ induced infiltration of inflammatory cells in the myocardial tissue in all groups and worsened the tissue damage and perivascular fibrosis in the AS+ISO group. Ultrastructural changes were observed in all TRZ-treated groups, including increased mitochondrial volume density (33±3%, 33±2%, 37±2% in WT, AS, and AS+ISO respectively; p<0.001 vs PBO in all the groups), and percentage of swollen mitochondria (21±3%; 21±3%; 27±4% in AS+ISO mice; p<0.001 in all cases) with higher value observed in AS+ISO+TRZ-group compared to the other TRZ-treated groups (p<0.05 for both). ACEi_BB therapy prevented the TRZ-induced decrease in LVEF in both groups (p<0.0001 vs the corresponding TRZ-treated group). In AS+ISO mice, the systolic function was significantly improved, with LVEF values reaching levels beyond those seen in the AS+ISO+PBO group (55±3% vs 50±2%, p<0.001). Both groups showed a restoration of the intervals of ventricular electrical activity, with a decrease in the duration of both QTc and JTc intervals compared to the TRZ-treated groups (p<0.0001). Additionally, an improvement in cardiomyocyte histopathological pattern and CSA was observed in both WT+TRZ+ACEi_BB (240±9 μm2 vs 265±18 μm2, p<0.05 vs WT+TRZ group) and AS+ISO+TRZ+ACEi_BB (250±7 μm2 vs 308±15 μm2, p<0.0001 vs AS+ISO+TRZ). In the latter group, cardioprotective therapy further reduced CSA to values even lower than those seen in AS+ISO+PBO (250±7 μm2 vs 284±16 μm2, p<0.01). Cardiac ultrastructure was preserved in both cases, with reduced mitochondria volume density and swollen mitochondria (p<0.0001 vs TRZ-treated group, for both). In AS+ISO+ mice, signs of activation of the lysosomal autophagic compartment were observed, with evidence of vacuoles containing damaged and dilated mitochondria.
Conclusions: Modelling cardiotoxicity in mice with pre-existing cardiac dysfunction is a valuable approach to investigate the cardiotoxic effects of TRZ and explore its relationship with pre-existing cardiac dysfunction. This approach revealed that TRZ therapy leads to systolic impairment, cardiomyocyte hypertrophy, and mitochondrial abnormalities. TRZ induced cardiac dysfunction and remodelling to a comparable extent across the groups. When TRZ cardiotoxicity acted on top of conditions of pre-existing cardiac dysfunction and/or tissue alteration, a worse cardiac phenotype was observed. Overall, these data provide an improved understanding of the extent of cellular and ultrastructural damage that result from TRZ treatment in mice. Our findings showed that captopril and bisoprolol protected against TRZ-induced cardiac remodelling and dysfunction. Notably, the beneficial the effect of captopril and bisoprolol was significant in mice with pre-existing cardiac impairment, providing valuable insights for future research on cardioprotection against TRZ-induced cardiotoxicity in the high-risk setting.
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