Remote Ischemic Preconditioning Ameliorates Anthracycline-induced Cardiotoxicity and Preserves Mitochondrial Integrity

Aims: Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect in a significant proportion of cancer patients. A central mechanism of AIC is irreversible mitochondrial damage. Despite major efforts, there are currently no effective therapies able to prevent AIC. Methods and Results: Forty Large-White pigs were included. In Study 1, 20 pigs were randomized 1:1 to remote ischemic pre-conditioning (RIPC, 3 cycles of 5 min leg ischemia followed by 5 min reperfusion) or no pretreatment. RIPC was performed immediately before each of five intracoronary doxorubicin injections (0.45 mg/kg) given at weeks 0, 2, 4, 6, and 8. A group of 10 pigs with no exposure to doxorubicin served as healthy controls. Pigs underwent serial cardiac magnetic resonance (CMR) exams at baseline and at weeks 6, 8, 12, and 16. After 16-week CMR, pigs were sacrificed and tissue samples collected. In study 2, 10 new pigs received 3 doxorubicin injections (with/out preceding RIPC) and were sacrificed 2 weeks after the third dose. In Study 1, LVEF remained unchanged in doxorubicin-treated pigs until week 6 (time of the fourth doxorubicin injection). From there on, LVEF progressively declined, but LVEF depression was blunted animals receiving RIPC before doxorubicin (RIPC-Doxo), which had a significantly higher LVEF at week 16 than doxorubicin treated pigs that received no pretreatment (Untreated-Doxo) (mean (SD) 41.5% (9.1) vs 32.5% (8.7), p=0.04). Preserved LVEF was mainly due to conserved contractile function, as evidenced by smaller LVESV, and better regional contractile function. In Study 2, transmission electron microscopy (TEM) after 3 doxorubicin doses showed fragmented mitochondria with severe morphological abnormalities in RIPC+Doxo pigs, together with upregulation of fission proteins and autophagy markers on western blot. At the end of the 16-week Study 1 protocol, TEM revealed overt mitochondrial fragmentation with structural fragmentation in Untreated-Doxo pigs, whereas interstitial fibrosis was significantly less severe in the RIPC+Doxo pigs. Conclusion: In a translatable large animal model of AIC, RIPC applied immediately before each doxorubicin injection resulted in preserved cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented mitochondrial fragmentation and dysregulated autophagy from the early stages of AIC. RIPC is a promising intervention for testing in clinical trials in AIC.

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Remote ischaemic preconditioning ameliorates anthracycline-induced cardiotoxicity and preserves mitochondrial integrity

Cardiovascular Research ◽

10.1093/cvr/cvaa181 ◽

2020 ◽

Author(s):

Carlos Galán-Arriola ◽

Rocio Villena-Gutiérrez ◽

María I Higuero-Verdejo ◽

Iván A Díaz-Rengifo ◽

Gonzalo Pizarro ◽

...

Keyword(s):

Contractile Function ◽

Interstitial Fibrosis ◽

Left Ventricular ◽

Large Animal Model ◽

Left Ventricular Ejection ◽

Large Animal ◽

Ischaemic Preconditioning ◽

Mitochondrial Fragmentation ◽

Ventricular Ejection Fraction ◽

Remote Ischaemic Preconditioning

Abstract Aims Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect among cancer patients. A central mechanism of AIC is irreversible mitochondrial damage. Despite major efforts, there are currently no effective therapies able to prevent AIC. Methods and results Forty Large-White pigs were included. In Study 1, 20 pigs were randomized 1:1 to remote ischaemic preconditioning (RIPC, 3 cycles of 5 min leg ischaemia followed by 5 min reperfusion) or no pretreatment. RIPC was performed immediately before each intracoronary doxorubicin injections (0.45 mg/kg) given at Weeks 0, 2, 4, 6, and 8. A group of 10 pigs with no exposure to doxorubicin served as healthy controls. Pigs underwent serial cardiac magnetic resonance (CMR) exams at baseline and at Weeks 6, 8, 12, and 16, being sacrifice after that. In Study 2, 10 new pigs received 3 doxorubicin injections (with/out preceding RIPC) and were sacrificed at week 6. In Study 1, left ventricular ejection fraction (LVEF) depression was blunted animals receiving RIPC before doxorubicin (RIPC-Doxo), which had a significantly higher LVEF at Week 16 than doxorubicin treated pigs that received no pretreatment (Untreated-Doxo) (41.5 ± 9.1% vs. 32.5 ± 8.7%, P = 0.04). It was mainly due to conserved regional contractile function. In Study 2, transmission electron microscopy (TEM) at Week 6 showed fragmented mitochondria with severe morphological abnormalities in Untreated-Doxo pigs, together with upregulation of fission and autophagy proteins. At the end of the 16-week Study 1 protocol, TEM revealed overt mitochondrial fragmentation with structural fragmentation in Untreated-Doxo pigs, whereas interstitial fibrosis was less severe in RIPC+Doxo pigs. Conclusion In a translatable large-animal model of AIC, RIPC applied immediately before each doxorubicin injection resulted in preserved cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented mitochondrial fragmentation and dysregulated autophagy from AIC early stages. RIPC is a promising intervention for testing in clinical trials in AIC.

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Abstract 2874: Bone Marrow-Derived Mononuclear Cell Transfer Is Associated with Superior Cardiac Recovery and Neovascularization as Compared to Nonmodified Mesenchymal Stem Cell Therapy in Chronic Myocardial Infarction

Circulation ◽

10.1161/circ.118.suppl_18.s_790 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Myrielle Mathieu ◽

Bachar El Oumeiri ◽

Karim Touihri ◽

Philippe Thoma ◽

Thierry Metens ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Systolic Function ◽

Cardiac Contractility ◽

Autologous Bone Marrow ◽

Functional Improvement ◽

Large Animal Model ◽

Controlled Study ◽

Large Animal ◽

Mesenchymal Stem Cell Therapy

Background: Cell type with optimal efficacy for cardiac repair remains under discussion. We conducted a randomized, blind and placebo-controlled study with head-to-head comparison of autologous bone marrow mononuclear cells (BMNC) and autologous non-modified mesenchymal stem cells (BMSC) therapy in a large animal model of chronic MI. Methods: Eleven weeks after coronary ligation, 24 dogs received intramyocardial injections of placebo (n = 8), BMNC (227.10 6 ± 32.10 6 cells, n = 8) or nonmodified-MSC (232.10 6 ± 40.10 6 cells, n = 8). Cardiac performance and remodelling were assessed up to 16 weeks follow-up (FU). Results: (Table ) BMNC injection induced a decrease in the WMSI at 8 and 16 weeks as compared to baseline (BL). In contrast, injection of the BMSC was associated with a moderate decrease at 16 weeks vs BL. Furthermore, LV end-systolic elastance (Ees) increased only after injection of BMNC and remained unchanged after BMSCs injection. Conclusions: In the canine model of chronic MI, myocardial BMNC transfer is superior to non-modified BMSC to reduce infarct size, improve regional systolic function and cardiac contractility. Functional improvement is associated with favourable angiogenic environment and neovascularization.

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Abstract P380: Common And Distinct Signatures Of Interstitial And Perivascular Fibrosis In Mouse Models Of Hypertensive Heart Disease

Circulation Research ◽

10.1161/res.129.suppl_1.p380 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Louise Thisted ◽

Claudia Correia ◽

Karin Jennbacken ◽

Maria Wagberg ◽

Franziska Wichern ◽

...

Keyword(s):

Heart Disease ◽

Mouse Models ◽

Vascular Function ◽

Cardiac Fibrosis ◽

Interstitial Fibrosis ◽

Severe Disease ◽

Cardiac Contractility ◽

Hypertensive Heart Disease ◽

Light Sheet ◽

Preclinical Research

Fibrosis is the hallmark of hypertensive heart disease and heart failure with preserved ejection fraction. Perivascular fibrosis impairs vascular function while interstitial fibrosis leads to compromised cardiac contractility. How these fibrosis types are represented in mouse models of hypertensive heart disease and to what extent the transcriptional signatures of cardiac fibrosis are defined by their location is unknown. Mice were dosed over 4 weeks with angiotensin II (AngII) alone or together with α 1 -adrenergic agonist phenylephrine (PE) and were characterized by echocardiography, light sheet imaging and fibrosis histology. While both groups developed systolic and diastolic dysfunction, hypertrophy and perivascular fibrosis, co-administration of PE resulted in a more severe disease phenotype and prevalent interstitial fibrosis, highlighting the benefits of this model in preclinical research. High-precision spatial transcriptomics based on laser capture microdissected perivascular and interstitial fibrotic areas revealed activation of distinct pro-fibrotic as well as cardioprotective pathways in the AngII+PE infusion model. Perivascular and interstitial fibrosis showed remarkable differences in global gene expression signatures, as demonstrated by high expression of osteochondrogenic genes and markers of secretory fibroblasts in perivascular fibrosis. A limited number of upregulated genes is shared between the fibrosis locations. These data collectively show the suitability of mouse models of hypertensive heart disease to study cardiac fibrosis and demonstrate how progression of fibrosis in mice is closely coupled to deteriorating cardiac dysfunction associated with highly distinct molecular signatures of perivascular and interstitial fibrosis.

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EphrinA1-Fc Attenuates Ventricular Remodeling and Dysfunction in Chronically Nonreperfused WT but not EphA2-R-M mice

International Journal of Molecular Sciences ◽

10.3390/ijms21165811 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5811

Author(s):

K’Shylah S. Whitehurst ◽

Victoria A. Chan ◽

Heather K. Estes ◽

Smrithi Valsaraj ◽

Susan Kent ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Ventricular Remodeling ◽

Contractile Function ◽

Interstitial Fibrosis ◽

Left Ventricular ◽

High Dose ◽

Coronary Artery Ligation ◽

Wild Type ◽

Western Blots ◽

Primary Mouse

Background: EphrinA1-Fc abolishes acute I/R injury and attenuates nonreperfused cardiac injury 4 days after permanent occlusion in mice. The goal of this study was to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of coronary artery ligation, to determine the degree to which early salvage effects translate to reduced adverse remodeling after 4 weeks of nonreperfused myocardial infarction (MI) in wild-type B6 and EphA2-R-M (EphA2 receptor null) mice. Methods: At 4 weeks post-MI, echocardiography, histologic and immunohistochemical analyses of B6 mouse hearts were performed. Primary mouse cardiac fibroblasts (FBs) isolated from B6 mice cultured in the presence of low and high dose ephrinA1-Fc, both with and without pro-fibrotic TGF-β stimulation and Western blots, were probed for relative expression of remodeling proteins MMP-2, MMP-9 and TIMP-1, in addition to DDR2 and (p)SMAD2/3/totalSMAD2/3. Results: EphrinA1-Fc preserved a significant degree of contractile function, decreased adverse left ventricular remodeling, attenuated excessive compensatory hypertrophy, and decreased interstitial fibrosis in wild-type (WT) B6 mouse hearts. In contrast, most of these parameters were poorer in ephrinA1-Fc-treated EphA2-R-M mice. Of note, fibrosis was proportionately decreased, implying that other EphA receptor(s) are more important in regulating the pro-fibrotic response. Primary FBs showed disparate alteration of MMP-2, MMP-9 and TIMP-1, as well as DDR2 and p-SMAD2/3/totalSMAD2/3, which indicates that matrix remodeling and cardiac fibrosis in the injured heart are influenced by ephrinA1-Fc. Conclusion: This study demonstrates the capacity of a single administration of ephrinA1-Fc at the onset of injury to attenuate long-term nonreperfused post-MI ventricular remodeling that results in progressive heart failure, and the important role of EphA2 in mitigating the deleterious effects.

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Remote ischemic preconditioning ameliorates anthracycline-induced cardiotoxicity and preserves mitochondrial integrity: results from a randomized preclinical trial in pigs

European Heart Journal ◽

10.1093/ehjci/ehaa946.3674 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

C Galan-Arriola ◽

R Villena-Gutierrez ◽

M.I Higuero-Verdejo ◽

I.A Diaz-Rengifo ◽

G Pizarro ◽

...

Keyword(s):

Animal Model ◽

Ischemic Preconditioning ◽

Contractile Function ◽

Left Ventricular ◽

Remote Ischemic Preconditioning ◽

Large Animal Model ◽

Left Ventricular Ejection ◽

Funding Source ◽

Large Animal ◽

Regional Contractile Function

Abstract Introduction Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect occurring in a significant proportion of patients. Irreversible mitochondrial damage is a central mechanism of AIC. Despite many efforts, there is a lack of therapies able to prevent AIC. Remote ischemic preconditioning (RIPC) could be a promising therapy to prevent AIC due to the scheduled application of chemotherapy in cancer patients. Purpose To evaluate the cardioprotective efficacy of RIPC in large animal model of AIC. Methods Large-White pigs (n=20) underwent a validated protocol of AIC consisting on five intracoronary doxorubicin injections (0.45 mg/kg), on weeks 0, 2, 4, 6, 8 of the study. Pigs were randomized before the initiation of the study to remote ischemic pre-conditioning (RIPC, 3 cycles of 5 min lower limb ischemia followed by 5 min reperfusion) or sham procedure immediately before doxorubicin injections. An additional group of 10 pigs without any exposure to doxorubicin was carried out as controls. Pigs underwent a comprehensive serial cardiac magnetic resonance (CMR) exam baseline, and on weeks 6, 8, 12, and 16. After 16-week CMR, pigs were sacrificed and tissue samples collected. A second group of 10 pigs (randomized 1:1 for RIPC) underwent the same protocol but were sacrificed 2 weeks after the third doxorubicin dose for early evaluation of tissue changes. Primary endpoint of the study was CMR-based left ventricular ejection fraction on week 16. Results Until week 6 (time of fourth doxorubicin injection), LVEF remained unchanged in both groups. From there on, a progressive decline in LVEF was observed. LVEF depression trajectory was blunted in RIPC animals. Compared to controls, pigs undergoing RIPC before each doxorubicin dose had a significantly higher LVEF at week 16: median (IQR) 45% (27–50%) vs 33% (19–47%) in RIPC and controls respectively, p=0.04. Improvement in LVEF was mainly due to a more preserved contractile function, as evidence by smaller LVESV, and better regional contractile function. After 3 doxorubicin doses, a time where global (LVEF) and regional contractile function was still unchanged, transmission electron microscopy (TEM) showed fragmented mitochondria with remodeled cristae only in control pigs. At the end of the 16 weeks, TEM evaluation in control pigs (as compared to RIPC pigs) showed overt cardiomyocyte's mitochondrial fragmentation with overt structural derangement. At this time, RIPC pigs had significantly less interstitial fibrosis on histology. Conclusions In a translatable large animal model of AIC, RIPC applied immediately before each doxorubicin cycle resulted in a preservation of cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented the deleterious effects of doxorubicin on mitochondria since early stages of AIC. RIPC is a promising intervention to be tested in clinical trials to prevent cardiotoxicity. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovaciόn and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)

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Association between Circular RNA CDR1as and Post-Infarction Cardiac Function in Pig Ischemic Heart Failure: Influence of the Anti-Fibrotic Natural Compounds Bufalin and Lycorine

Biomolecules ◽

10.3390/biom10081180 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1180

Author(s):

Julia Mester-Tonczar ◽

Johannes Winkler ◽

Patrick Einzinger ◽

Ena Hasimbegovic ◽

Nina Kastner ◽

...

Keyword(s):

Heart Failure ◽

Animal Model ◽

Cardiac Fibrosis ◽

Circular Rna ◽

Natural Compounds ◽

Tissue Level ◽

Large Animal Model ◽

Circular Rnas ◽

Large Animal

Anti-fibrotic therapies are of increasing interest to combat cardiac remodeling and heart failure progression. Recently, anti-fibrotic circular RNAs (circRNAs) have been identified in human and rodent cardiac tissue. In vivo (rodent) experiments proved cardiac anti-fibrotic effects of the natural compounds bufalin and lycorine by downregulating miRNA-671-5p, associated with a theoretic increase in the tissue level of circRNA CDR1as. Accordingly, we hypothesized that both anti-fibrotic drugs may inhibit focal myocardial fibrosis of the remodeled left ventricle (LV) also in a translational large animal model of heart failure (HF). Domestic pigs were repeatedly treated with subcutaneous injections of either bufalin, lycorine, or saline, (n = 5/group) between days 7–21 post acute myocardial infarction (AMI). At the 2-month follow-up, both bufalin and lycorine led to significantly reduced cardiac fibrosis. Bufalin treatment additionally led to smaller end-diastolic volumes, higher LV ejection fraction (EF), and increased expression of CDR1as of the AMI region. Elevated tissue levels of the circRNA CDR1as in the AMI region of the pig heart correlated significantly with LV and right ventricular EF, LV stroke volume, and negatively with infarct size. In conclusion, we successfully identified the circRNA CDR1as in pig hearts and show a significant association with improved LV and RV function by anti-fibrotic therapies in a translational animal model of HF.

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TOTAL SPINAL BLOCKADE WITH INTRATHECAL BUPIVICAINE PRESERVES CARDIAC CONTRACTILITY IN A LARGE ANIMAL MODEL OF BRAINSTEM DEATH

Transplantation Journal ◽

10.1097/00007890-201007272-00494 ◽

2010 ◽

Vol 90 ◽

pp. 262

Author(s):

W. A. Almoustadi ◽

T. W.R. Lee ◽

B. Xiang ◽

R. C. Arora ◽

H. Lin ◽

...

Keyword(s):

Animal Model ◽

Cardiac Contractility ◽

Large Animal Model ◽

Large Animal ◽

Brainstem Death

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Abstract 200: Cardiomyocyte-specific Deletion Of Rhoa Accelerates Heart Failure But Prevents Cardiac Fibrosis In Response To Pressure-overload

Circulation Research ◽

10.1161/res.113.suppl_1.a200 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Jessica Lauriol ◽

Kimberly Keith ◽

Sanjeewa A Goonasekera ◽

Qingen Ke ◽

Peter Kang ◽

...

Keyword(s):

Heart Failure ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Acute Stress ◽

Contractile Function ◽

Interstitial Fibrosis ◽

Growth Parameters ◽

Specific Deletion

Heart failure is a progressive disease characterized by loss of cardiac contractility, interstitial fibrosis, and chamber remodeling. Though several signaling pathways have been identified, the precise mechanisms involved in regulating pathological cardiac responses remain unclear. The Ras-related small G protein RhoA is an identified mediator of in vivo pathological hypertrophy. However, it also promotes cell survival and is cardioprotective after ischemia/repurfusion injury. Therefore, to determine the molecular mechanisms that underlie these opposing roles for RhoA in the myocardium, we generated mice with cardiomyocyte-specific deletion of RhoA (RhoAfl/fl-αMHC-Cre). As compared to wildtype mice, hearts from RhoAfl/fl-αMHC-Cre mice showed similar functional, structural, and growth parameters, both at baseline and in response to acute transverse aortic constriction (TAC) (2wks), suggesting RhoA plays no major role in either homeostatic or acute stress-induced cardiac conditions. However, after exposure to chronic TAC for 8wks, hearts from RhoAfl/fl-αMHC-Cre mice developed an accelerated dilation, with significant loss of contractile function. Mechanistically, cardiomyocyte-specific loss of RhoA reduced ERK1/2 activation and perturbed agonist-evoked calcium cycling, through aberrant IP3 and DAG signaling, indicating that RhoA mediates its cardioprotective effects through regulation of these pathways. Despite this, and surprisingly, hearts from RhoAfl/fl-αMHC mice also showed significantly decreased TAC-induced cardiac fibrosis, with a demonstrated decrease in transcriptional activation of genes involved in the fibrotic response, including the serum response factor (SRF), as well as the secreted factor BMP7. Taken together, our data reveal an interesting, yet highly complex signaling and regulatory mechanism for RhoA, whereby RhoA signaling is both cardioprotective against development of heart failure in response to chronic stress, but also cardio-deleterious through its capacity to activate pro-fibrotic genes.

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