Pazopanib for Renal Cell Carcinoma Leads to Elevated Mean Arterial Pressures in a Murine Model

Abstract

Background:

In the setting of metastatic RCC (mRCC), pazopanib is approved as first line therapy. Unfortunately treatment may lead to cardiotoxicity such as hypertension, heart failure, and myocardial ischemia.

Objective:

Define the in vivo role of pazopanib in the development of cardiotoxicity.

Methods:

Wild type mice were dosed for 42 days via oral gavage, and separated into control and treatment (pazopanib) groups. Baseline ECG’s, echocardiograms, and blood pressures were recorded. At the conclusion of the study functional parameters were again recorded, and animals were used for pathological, histological, and protein analysis.

Results:

After 2 weeks of dosing with pazopanib, the treatment group exhibited a statistically significant increase in mean arterial pressure compared to control mice (119 + 11.7 mmHg versus 108 + 8.2 mmHg, p=0.049). Treatment with pazopanib led to a significant reduction in the cardiac output of mice.

Conclusion:

Our findings in mice clearly demonstrate that treatment with pazopanib leads to a significant elevation in blood pressure after 2 weeks of dosing and this persists for the duration of dosing. The continued development of the cardio-oncology field will be paramount in providing optimal oncologic care while simultaneously improving cardiac outcomes through further investigation into the mechanisms of CV toxicity.

Introduction

Renal cell carcinoma (RCC) is the 6th most common cancer in the U.S (and most common kidney cancer) and is associated with a high number of average years of life lost (16 years)^1,2. In the setting of metastatic RCC (mRCC), five tyrosine kinase inhibitors (TKIs) of the vascular endothelial growth factor receptor (VEGFR) pathway are approved by the Food and Drug Administration (FDA), and pazopanib is approved as first line therapy for RCC³.

Angiogenesis is critical in the pathogenesis of RCC, and pazopanib leads to tumor regression and inhibition of angiogenesis by inhibiting VEGFR 1–3⁴. Platelet-derived growth factor receptors and stem cell factor KIT receptors are also inhibited by pazopanib. Unfortunately there are many off-target effects with this therapy, and pazopanib has been associated with diarrhea, fatigue, hair depigmentation, nausea, and elevated liver function enzymes³. Furthermore, cardiotoxicity such as hypertension (HTN), heart failure (HF) with a reduced left ventricular ejection fraction (LVEF), and myocardial ischemia (elevated cardiac troponin) have been reported in up to half of all patients on pazopanib^5–7. Of particular concern is that it has been reported that the incidence of HTN with pazopanib is as high as 40%, with 26% of patients having elevated N-terminal B-type natriuretic peptide (NT-pro-BNP) levels, and 7% with a reduction in their LVEF⁵. Notably, in a meta-analysis of randomized control trials with >10,000 patients, the incidence of HF related to VEGFR TKIs 2.4%⁸. Another large meta-analysis reported that the overall incidence of all-grade HF associated with VEGFR-TKIs was 3.2%⁹. With a median age of 60 years at RCC diagnosis, older patients with cardiovascular (CV) risk factors may be exceptionally vulnerable to adverse events. A clinical need exists to identify TKI-induced early molecular alterations in the heart prior to the onset of HTN, HF, and arrhythmias, which limits the efficacy and deprives cancer patients of life extending therapies.

The goal of our study was to define the in vivo role of pazopanib in the development of HTN and HF. Using a murine model to investigate the CV effects of pazopanib, our data demonstrates that wild type (WT) mice treated with pazopanib for 6 weeks develop HTN early in the course of treatment (by week 2) and the effect is sustained throughout dosing. We also demonstrate that treatment with pazopanib leads to a significant reduction in the cardiac output (CO) of mice.

Methods

Animals

C57BL/6 black mice (all animals were 8 weeks of age at the start of dosing) were dosed for 42 days via oral gavage by a trained and experienced technician. All experiments were approved by the institutional animal care and use committees and were performed in accordance with all National Institutes of Health guidelines for the humane treatment of animals.

There were 8 control and 8 treatment animals at the beginning of the study (n=16). Experimental mice received pazopanib (treatment group), and control mice received 2.5% DMSO only (control group). Pazopanib HCl (Selleckchem.com) was dissolved in 100% sterile DMSO (Sigma-Aldrich) and then diluted down to 2.5% DMSO with DEPC (GeneMate). Both drugs were administered to mice via oral gavage, and dosing was performed twice daily. Pazopanib dosing was 30 mg/kg twice daily as previously described¹⁰. After 1 week of dosing two animals died (one control and one treatment). An autopsy was performed on each animal and it was determined (by 3 separate investigators) that the cause of death was iatrogenic in nature (Air bubble in the stomach due to a faulty gavage needle, which was subsequently replaced). The rest of the animals (n=14) survived until the conclusion of the study. Animals were subsequently randomly assigned to 3 groups: 1) Four animals (2 experimental and 2 control) had their hearts and kidneys removed, and then placed into 10% formalin for pathological analysis; 2) Four animals, (2 experimental and 2 control) were used for patch clamping to obtain preliminary electrophysiological data; 3) Six animals (3 experimental and 3 control) were designated to have their heart, lungs, liver, kidneys, and brain removed for the purpose of making protein lysate for immunoblot analysis. Sections of hearts were saved from each animal and stored in a −80°C freezer for future RNA work.

Organ Removal/Blood Collection

All animals were injected with heparin (100 units/kg, Fresenius Kabi USA LLC) ~20 minutes before organ removal and then anesthetized with isoflurane (Henry Schein). The mice were anesthetized using 2.5 % isoflurane in 95% O₂ / 5% CO₂ at a rate of 1 L/min; this level was maintained upon moving the animals to the nose cone. A pain test was performed and once no response was seen the animals which were to be used for lysate and pathology had their eye(s) removed in order to collect serum samples. The blood was left sitting on lab top for ~20 minutes to allow clotting and then spun at 4°C for 15 minutes at 3000 rpm. The serum was collected and stored at −80°C. After the pain test, all animals had their heart, lungs, liver, kidneys, and brain removed and washed in PBS. Hearts that were used for ventricular cannulation were not weighed. After the PBS wash the organs were placed on a kimwipe (Kimtech Science) to soak up excess PBS and then placed into pre-weighed weigh boats. Weights were collected and organs were either placed into 10% formalin for pathology, or placed on dry ice for storage or to be used for lysates.

Weights

Animals were weighed every other day during the first week and twice weekly thereafter. Animals were removed from their home cage one at a time and placed into a clear plastic container. The container, which was previously zeroed and balanced, was placed onto a Mettler Toledo new classic ML 1502E scale for weighing. The weight was then recorded when the animal was sitting still on the scale. Weights were recorded to the first decimal place. These weights were then uses to dose the animals.

Dose Preparation

Once weights were obtained they were plugged into a formatted excel sheet designed to calculate the amount of Pazopanib or DMSO required for dosing. The pazopanib was made the evening prior to dosing and was only used for a maximum of 4 days. The pazopanib was weighed on a Mettler Toledo AL104 and was weighed out to the ten thousandths place. The appropriate amount of DMSO (as determined by the excel sheet) was added to dissolve the pazopanib. The mix was then diluted to a 2.5% concentration of DMSO with pre-filtered DECP. The mixture was wrapped in tinfoil and kept at room temperature.

Oral Gavage

A 20 gauge 38cm ball tip cannula was used to administer drug to animals. Mice were scuffed according to standard animal care operating procedures. The mice were scruffed by grasping the skin over the shoulders with the thumb and middle fingers. Next the head was gently extended back with the index finger placed on top of the head to create a straight line through the neck and esophagus. While holding the animal parallel to the work surface the gavage needle was placed in either side of the mouth, over the tongue, and advanced through the pharynx by gently pressing the gavage cannula to the roof of the mouth to elevate the head and straighten the esophagus. If there was any resistance the gavage cannula was not forced and another attempt was made after completely pulling the cannula out. After dosing the cannula was gently removed following the same angle as insertion. The mice were then returned to their cage and monitored for 5–10 minutes, assessing for any sign of labored breathing or distress. Control animals were always dosed prior to treatment animals and with different cannulas. Cannulas were cleaned with double distilled water, followed by ethanol, and then were dried with kimwipes after each dose.

Blood Pressure Measurement

The CODA Non-Invasive Blood Pressure System was used for recordings (Kent Scientific Corporation, Torrington, CT, USA). Mice were individually removed from their cage and then were held by their tail to allow them to enter the blood pressure tube on their own accord. The O-cuff was then placed as close to the base of their tail as possible or until resistance was felt. The VPR-cuff was placed against the O-cuff. Animals had blood pressure (BP) recordings twice per week for two weeks prior to dosing (acclimation) and then once a week throughout the course of the study, with one additional week after the conclusion of dosing (total 9 weeks). During the acclimation phase each animal received 20 acclimation cycles, and these recordings were not used for analysis. For all weeks except the acclamation phase if an animal received <3 measureable cycles after the 10 measured cycles the animal was returned to its home cage for one hour and then another attempt was made to obtain recordings. Cycles which were determined useable were then averaged together per animal for analysis. At the conclusion of the study three experimental mice and three control mice had final BP recordings after drug wash-out to assess for sustained drug effects on the cardiovascular system.

Immunoblots

Tissue was harvested from 3 control and 3 treatment animals, and then immediately placed on dry ice. The samples were then immediately placed in a −80°C freezer until all hearts had been collected. To create lysates, hearts were placed into ice cold Homogenization Buffer (in mM: 50 Tris-HCl, 10 NaCl, 320 sucrose, 5 EDTA, 2.5 EGTA; supplemented with 1:1000 protease inhibitor cocktail and 1:500 PMSF). Following BCA quantitation, tissue lysates were electrophoresed using the Mini-PROTEAN tetra cell (BioRad) and a 4–15% precast TGX gel (BioRad). Gels were transferred to a nitrocellulose membrane using the Mini-PROTEAN tetra cell (BioRad) and blocked for 1 hour at room temperature. Primary antibody incubation was carried out overnight at 4 °C. Densitometric analyses were performed using ImageLab software (BioRad). For all experiments, protein values were normalized against an internal loading control validated against the specific pathology.

Antibodies

The following antibodies were used: Angiotensin II Type 1 and 2 (ABCAM, Rabbit, monoclonal), VEGFA (ABCAM, mouse monoclonal), VEGF-Rec (ABCAM, rabbit polyclonal), and FGF2 (Millipore, mouse monoclonal).

Echocardiography

Once weekly starting from one week prior to dosing (baseline) and going until one week after dosing (total 8 weeks), 14 animals (7 control animals and 7 treatment animals) had echocardiograms performed. Digital images were obtained at a frame rate of 180 images/s. Transthoracic echocardiogram was performed using the Vevo 2100 (Visualsonics). The mice were anesthetized using 2.0 % isoflurane in 95% O₂ / 5% CO₂ at a rate of 1 L/min. Anesthesia was maintained by administration of oxygen and 1.5% isoflurane. Electrode gel was placed on the ECG sensors of the heated platform and the mouse was placed supine on the platform to monitor electrical activity of heart. The MS-400 transducer was used to collect the contractile parameters of the heart in the short axis M-mode. LV wall thickness [interventricular septum (IVS) and posterior wall (PW) thickness] and internal dimensions at diastole and systole (LVIDd and LVIDs, respectively) were measured. LV fractional shortening [(LVIDd - LVIDs)/LVIDd], relative wall thickness [(IVS thickness + PW thickness)/LVIDd], and LV mass [1.05 (IVS thickness + LVIDd + PW thickness) 3 - LVIDd3] were calculated from the M-mode measurements.

Surface Electrocardiography

Once a week starting from one week prior to dosing (baseline) and going until one week after dosing (total 8 weeks), 14 animals (7 control animals and 7 treatment animals) had surface ECG’s performed. Surface ECG recordings were obtained using 3 leads to measure heart activity within a 5-minute time frame. The mice were anesthetized using 2.0 % isoflurane in 95% O₂ / 5% CO₂ at a rate of 1 L/min. Anesthesia was maintained by administration of oxygen and 1.5% isoflurane. Heart rate, QRS duration, QTc and QT interval were automatically calculated by Lab Chart 7 using the ECG tracings.

Statistical Analysis

Continuous variables are presented as means with standard deviations. Categorical variables were compared using Student’s t-test, or chi-square test, as appropriate. Two-way ANOVA was used to compare the mean difference in blood pressure between groups split on two independent variables (change in blood pressure over length of study/time). For all comparisons, P < 0.05 was considered significant. All statistical analyses were performed using STATA 14 (College Station, Texas 77845–4512).

Results

I. Impact of Pazopanib on growth and body weight

To determine the effects of pazopanib on cardiac function and blood pressure, we treated 8 week old C57BL/6 mice with pazopanib at 30 mg/kg per twice daily or DMSO as a control. In vivo studies have demonstrated that pazopanib inhibits the growth of a broad range of human tumor xenografts in mice, and a single oral dose of 30 mg/kg inhibits VEGF-induced VEGFR2 phosphorylation¹⁰. Notably, these pharmacokinetic studies showed that a pazopanib concentration of ≥40 μmol/L is required for inhibition of VEGFR2 in mice, and this was achieved with 30 mg/kg dosing. In patients a target steady-state concentration of ≥40 μmol/L was achieved in the majority of patients receiving doses of ≥800 mg once daily¹¹. Pazopanib or DMSO was administered for 42 days, twice the duration of time in the original pharmacokinetic studies¹⁰ but consistent with standard clinical practice.

In order to assess if pazopanib had an impact on normal aging and weight gain in experimental mice, body weights were recorded weekly in control and treatment animals (n=7 for both groups). Over the course of the 6 week study both groups of animals exhibited a consistent increase in body weight, and there was no significant difference in the mean weight between the groups at the completion of dosing (Figure 1; Control 22.79 + 0.77 and Treatment 22.78 + 1.00). Additionally, at the completion of the study there was no significant difference between the groups with respect to heart weight, tibia length, heart/tibia length, kidney weight, liver weight or brain weight (Table 1).

Figure 1. Animal weight over course of dosing.

A). Mice were weighed once a week over the course of the study. No significant differences were seen in body weights between control/treatment animals (n=7/7). B). Animals were consistently dosed twice a day at 9 am + 1 hour and 3:30 pm + 1 hour.

Table 1.

Body/organ measurements in control and treatment (pazopanib) mice at the conclusion of drug treatment.

Measurements in Male Mice at Conclusion of Dosing
	HW (g)	HW/TL (g/mm)	Lung W (g)	KW (g)	LIVER W (g)	BW (g)	TL (mm)
Control average	0.105 ± 0.0308	0.00602 ± 0.00180	0.134 ± 0.0214	0.292 ± 0.0171	0.923 ± 0.0695	0.427 ± 0.0293	17.3 ± 0.516
Treatment average	0.110 ± 0.00794	0.00638 ± 0.000437	0.135 ± 0.0130	0.254 ± 0.0535	0.918 ± 0.120	0.432 ± 0.0135	17.2 ± 0.393
P. Value	0.700495218	0.673690469	0.966929167	0.122624378	0.933943774	0.714647141	0.646233433

Values are means +/− SD for n of 7 mice per group. HW, Heart Weight; W, Weight; KW, Kidney Weight; BW, Brain Weight; and TL, Tibial Length.

II. Pazopanib Leads to Elevated Mean Arterial Pressure in Mice

Tail cuff blood pressure recordings were measured at the beginning of the study prior to dosing and after acclimation (baseline), and then once a week for 6 weeks in both groups. At baseline control mice had a mean arterial pressure (MAP) of 117 + 7.3 mmHg and treatment mice had a MAP of 114 + 8.9 mmHg (p=0.885, Figure 2). After 2 weeks of dosing with pazopanib, the treatment group exhibited a statistically significant increase in MAP compared to control mice (119 +11.7 mmHg versus 108 + 8.2 mmHg, p=0.049, Figure 2). Notably by the end of the study treatment mice continued to exhibit a statistically significant increase in MAP compared to controls (118 + 7.5 mmHg vs 107 + 3.5 mmHg, p=0.0042, Figure 2). Together these data demonstrate a significant increase of the MAP in treatment animals due to pazopanib with no significant change in pressure due to duration of the study (time).

Figure 2. Mean Arterial Pressure Over Time with Pazopanib Treatment.

Two-way ANOVA shows a significant increase of the mean arterial pressure in treatment animals due to drug with no significant change in pressure due to time (p=0.007). Black line represents treatment mice and red line represents control mice. The effect of the drug over time was also found to be significant (p=0.034). n=7 for treatment and control. * indicates significant change from controls at a given time point.

III. Effect of Pazopanib on Structural and Electrical Function

Echocardiograms and ECGs were performed on both groups of mice. Baseline and weekly measurements were made with both modalities. Regarding echocardiographic measurements, there was no significant difference between treatment and control groups with respect to left ventricular ejection fraction, stroke volume, systolic and diastolic diameters, and fractional shortening (Table 2). Of note, there was a statistically significant difference in cardiac output (CO) reflected by a decreased in CO in treatment mice compared to control mice (CO 16.6 + 0.9 L/min in control mice versus 13.9 + 0.83 L/min in the treatment group, p=0.047). To further assess structural changes at the organ level, heart and kidneys were removed from control and treatment mice and sent to pathology for histological analysis (n=2 in each group). There were no significant pathological changes observed between the groups (Figure 3). Regarding ECGs, heart rate, QRS, QT, and QTc were measured and there were no significant differences between the groups at any time interval.

Table 2.

Echocardiographic comparison between control and treatment (pazopanib) mice at baseline and after six weeks of treatment.

Table 2	LVEF (%)		Cardiac Output (mL/min)		Stroke Volume (μl)		Diastolic Diameter (mm)		Systolic Diameter (mm)		Fractional Shortening (%)
	Baseline	6 Weeks	Baseline	6 Weeks	Baseline	6 Weeks	Baseline	6 Weeks	Baseline	6 Weeks	Baseline	6 Weeks
Control	59.6 ± 2.10	63.2 ± 1.94	13.8 ± 1.09	16.6 ± 0.922	29.9 ± 1.72	37.6 ± 1.59	3.48 ± 0.121	3.73 ± 0.0756	2.41 ± 0.124	2.47 ± 0.0853	31.0 ± 1.37	33.7 ± 1.32
Treatment	56.4 ± 2.47	59.2 ± 0.491	15.3 ± 0.600	13.9 ± 0.829	33.8 ± 1.44	35.3 ± 1.07	3.74 ± 0.0414	3.74 ± 0.0449	2.65 ± 0.0775	2.58 ± 0.0313	29.1 ± 1.66	30.8 ± 0.342
P value	0.332	0.088	0.422	0.047	0.0965	0.243	0.195	0.991	0.437	0.269	0.863	0.0537

Values are means +/− standard error. n=6 for control baseline; n=7 control week 6; n= 7 treatment baseline and week 6.

Figure 3. Histological samples of heart and kidney sections between control and treatment mice.

A. (Heart) There were no over differences in ventricular wall thickness and luminal diameter via hematoxylin and eosin on the left and no overt differences in collagen via Trichrome staining on the right between control and pazopanib treated mice (n=2). B. (Kidney) There were no over differences in kidney sections via hematoxylin and eosin on the left and no overt differences in collagen via Trichrome staining on the right. By Masson’s trichrome staining, collagen is present in normal amounts in the interstitium around blood vessels in both the control group and Pazopanib treated mice (n=2).

IV. Effect of Pazopanib on Protein Expression

Immunoblots were performed on heart and kidney lysates to assess protein expression relevant to pathways which could be impacted by VEFG inhibition. Specifically we tested antibodies to VEGF-A, VEGF-receptor, Angiotensin Type I and Type II receptors, and Fibroblast Growth Factor-2 (FGF2). When comparing control and treatment animals, there were no significant differences in the protein expression for any of the antibodies in heart or kidney (Figure 4–5).

Figure 4. Protein expression from whole heart lysates.

Immunoblots were tested for VEGF-A, VEGF-receptor, Angiotensin Type I and Type II receptors, and Fibroblast Growth Factor-2 (FGF2) (A-E), with total protein serving as a loading control (F). When comparing control and treatment animals, there were no significant differences in the protein expression for any of the antibodies in heart.

Figure 5. Protein expression from whole kidney lysates.

Immunoblots were tested for VEGF-A, VEGF-receptor, Angiotensin Type I and Type II receptors, and Fibroblast Growth Factor-2 (FGF2) (A-E), with total protein serving as a loading control (F). When comparing control and treatment animals, there were no significant differences in the protein expression for any of the antibodies in kidney.

Discussion

Our findings in mice clearly demonstrate that treatment with pazopanib leads to a significant elevation in blood pressure after just 2 weeks of dosing and this persists for the duration of dosing. Notably we also observed a decrease in CO in mice treated with pazopanib, suggestive of early cardiomyocyte stress and possible remodeling in the presence of elevated MAPs. Previous data has demonstrated that mice can be treated with pazopanib safely at the doses used in our study, and pazopanib does not lead to decreased survival or negatively impact growth¹⁰. These data are important in that it confirms the hypertensive clinical phenotype often observed in human populations treated with pazopanib. Additionally these data suggest this animal model can be used to gain more insight into the mechanisms leading to pazopanib induced cardiotoxicity.

VEGF signaling pathway inhibitors (VSPI) have known efficacy in multiple malignancies by inhibiting tumor angiogenesis by direct and indirect pathways, but are increasingly being recognized as having cardiotoxic side effects. Small-molecule targeted VEGFR TKIs have significantly improved outcomes in advanced/mRCC as evidenced by 6 and 14 month increases in median progression free survival and overall survival, respectively, with sunitinib compared to earlier first line agents for mRCC^12,13. Pazopanib is a non-selective inhibitor of angiogenesis, and it has been demonstrated to target VEGFR-1, −2, and −3, platelet-derived growth factor receptor (PDGFR)-α and -β, and c-KIT^14–16. Pazopanib’s favorable side effect profile and cost-effectiveness have made it an appealing option for physicians and patients ^17–21, which suggests that the number of patients with mRCC treated with pazopanib will only increase, especially with the significant growth of an already aging population.

Pazopanib has been associated with a CV toxicity profile that includes arterial HTN, ischemic and thrombotic events, cardiomyopathy, and cardiac dysrhythmias^22–26. Notably, HTN is by far the most common side effect with a reported 35.9% incidence among pazopanib-treated patients²⁷. The incidence of HF may range from 1–6%, and the incidence of a significant reduction (15%) of absolute LVEF has been reported to be 9%^3,9. While none of the mice treated with pazopanib in our study developed signs of HF, they did demonstrate signs of early CV stress, which may represent a pre-HF signal. Notably all 7 animals in our study treated with pazopanib had an increase in their blood pressure, individually and as a group (Figure 2). Pazopanib-related conduction disturbances have been reported in animal and clinical studies. In rats, the calcium channel blocker diltiazem and the beta-blocker metoprolol were able to prevent pazopanib-induced QT prolongation²⁸. Two additional clinical meta-analyses suggested that TKIs as a class significantly increase the QT interval^29,30. In our study with mice we did not see a significant increase in the QT interval in mice treated with pazopanib. The consistently elevated BP in our study in mice treated with pazopanib is striking because in another study in mice treated with sunitinib (another TKI used in RCC) a significant rise in MAP was not observed³¹.

The lack of ECG or structural changes seen in our study may be explained by the following: 1) in mice β-MyHC is the predominant isoform in the ventricles of humans where in mice the α-MyHC isoform predominates, therefore mice may be better equipped to withstand external and internal stress³², 2) the duration of our study was too short, or 3) the mice required a stressor (catecholamines, surgically induced HF) in order to establish a structural heart disease phenotype. In an elegant study of mice treated with sunitinib, mice did not develop cardiomyocyte apoptosis until an increase in blood pressure was induced via transaortic constriction (TAC)³¹, and TAC led to an accelerated reduction in cardiac function, reductions in coronary flow reserve, and enhanced cardiac fibrosis compared with vehicle-treated mice³¹.

While these data with pazopanib are intriguing, we have not defined a mechanism for the elevation in BP or HTN at this time, which limits the translatability of these findings. There are several attractive hypotheses which have been proposed and will be explored in future mechanistic studies³³. Some of the proposed pathways leading to TKI-induced HTN include: 1) Dysregulation of vasoconstrictors and vasodilators with VEGF inhibition^34–37 2) Elevated systemic afterload after VEGF inhibition and destruction of endothelial cells³⁸ 3) Decrease survival of mesangial cells and altered glomerular function and filtration after inhibition of renal VEGF receptors³⁹, and 4) Soluble fms-like tyrosine kinase receptors may abolish VEGF signaling, further exacerbating the effects of VEGF inhibition by TKIs leading to HTN and renal dysfunction via thrombotic microangiopathy^40–42.

Additionally it will be important for us to use older mice in future studies. In aged (450 days old) mice treated with the TKI imatinib mesylate, GATA4 haploinsufficient mice were more susceptible to mitochondrial impairment and cell death⁴³. Humans treated with pazopanib tend to be older (mean age >60) and have multiple comorbidities at baseline, increasing their susceptibility to cardiotoxic side effects from TKIs⁵. Furthermore, we will stress the mice to further assess a phenotype (treadmill and catecholamine injections), and perform basic RNA transcriptional analyses to assess unique pathways upregulated via pazopanib.

Preexisting CV disease has been identified in as many as 35% of RCC patients in the US⁴⁴. Combined with the fact the cancer survivorship continues to improve with the rapid evolution of targeted therapies such as pazopanib, the intersection between cardiovascular and oncologic disease will undoubtedly continue to expand. Our findings illustrate that pazopanib leads to elevated MAPs in mice, consistent with clinical findings in humans. Thus, the continued development of the cardio-oncology field will be paramount in providing optimal oncologic care while simultaneously improving cardiac outcomes through further investigation into the mechanisms of CV toxicity.

Table 3.

Electrocardiographic comparison between control and treatment (pazopanib) mice at baseline and after six weeks of treatment.

Table 3	Heart Rate (BPM)		QRS (ms)		QT (ms)		QTc (ms)
	Baseline	Week 6	Baseline	Week 6	Baseline	Week 6	Baseline	Week 6
Control	415 ± 11.5	466 ± 23.6	8.33 ± 0.171	8.29 ± 0.222	15.4 ± 0.198	17.5 ± 0.506	40.5 ± 0.802	48.9 ± 2.54
Treatment	402 ± 16.3	448 ± 34.5	7.97 ± 0.177	7.95 ± 0.126	15.2 ± 0.591	17.7 ± 0.609	39.3 ± 2.13	48.6 ± 3.23
P-value	0.526	0.686	0.144	0.208	0.729	0.75	0.583	0.956

Values are means +/− standard error. n=7 Control and Treatment