Evaluating the Effects of Exercise and Phellodendron Amurense on Cardiac Function in a Prostate Cancer Mouse Model

Abstract

Introduction:

Cytotoxic treatments pose a significant cardiotoxic risk to men with prostate cancer (PCa). Exercise has been found to prevent cardiotoxicities. Previous work from our group has also identified the chemopreventative effects of phellodendron amurense (PhAm). We therefore aimed to compare the effectiveness of exercise ± PhAm on cardiac function in a transgenic PCa mouse model.

Methods:

A 12-week, 4 arm, randomized controlled study was performed. Twenty-four transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were randomly assigned to either the control, exercise, PhAm or ExPhAm treatment groups. Mice assigned to exercise were given continuous access to a running wheel. PhAm groups consumed a diet enriched with PhAm at 600 mg/kg. Control animals maintained a normal diet and activity. Pre-post echocardiography was performed to measure heart rate, interventricular septum (IVS) thickness, left ventricle (LV) internal diameter, LV volume, ejection fraction (EF), fractional shortening (FS), and left ventricle mass (LVM).

Results:

Exercise, PhAm and the combination were able to protect against an increase in end diastolic and end systolic LV mass (p=0.003; p=0.006, respectively). Though not significant, the LV/body mass was markedly higher in the exercise group (+4.5%) and combination (+1.1%) compared to the PhAm (−4.2%) and control groups (−10.3%). Exercise and ExPhAm also protected against increases in IVS thickening while an increase was found in both the PhAm and control groups (p<0.05).

Conclusion:

The results of our study suggest that exercise is the driving factor in promoting cardiac health in PCa and can prevent cardiotoxicities associated with the tumor burden.

New & Noteworthy:

Exercise is effective in preventing significant left ventricular hypertrophy (when evaluated using relative wall thickness) and promoting a younger mouse phenotype. Exercise was the driving force in the Ex and ExPhAm groups. This resulted in the prevention of significant left ventricular hypertrophy and promotion of a younger mouse phenotype.

Graphical Abstract

INTRODUCTION

Prostate cancer (PCa) is the second leading cause of cancer death in men in the US and the incidence rate has been increasing by 3% each year since 2014, according to the American Cancer Society [1,2]. However, the overall number of deaths due to PCa are low due to earlier detection and increased availability in treatment options. This leaves an increasing number of men, estimated to be about 3.1 million in the US, surviving PCa at risk for the long term consequences of treatment [1]. For this reason, it is imperative to gain a better understanding of the long-term impact of PCa and the various treatment modalities available.

Many existing treatment options for PCa pose a significant risk of cardiovascular disease (CVD), which often have a higher mortality rate than the course of the cancer itself [3]. Forty-five percent of the deaths among those with PCa is attributable to causes other than the cancer itself, among which CVD is the second highest cause [3]. The risks associated with this pre-existing comorbidity is only compounded with the increased likelihood of CVD in patients who are being treated with oncological therapy. Current research hypothesizes that common PCa therapies such as androgen deprivation therapy, second-generation androgen receptor blockers, androgen metabolism inhibitors, chemotherapy, and immunotherapy may cause ischemic heart disease, hypertension, heart failure, and QT interval prolongation [3].

Based on data from other conditions, exercise may work to prevent the cardiotoxic effects of cancer and its treatment in men with PCa [4–7]. In healthy patients, long term exercise has been found to increase ventricular wall thickness and increase contractile forces to compensate for an increased demand for blood supply [4]. This increase in cardiac myocyte size may also protect against any abnormalities that may arise in the heart’s conduction system by upregulating both depolarization and repolarization of cardiac myocytes [4]. Additionally, long term exercise protects against oxidative damage through altering mitochondrial protein expression [5]. These cardioprotective effects may also be seen in patients with cancer. A study found that breast cancer patients who had a high level of physical activity prior to their diagnosis had a lower overall risk of cardiovascular events compared to those who were not as physically active prior to diagnosis [6]. These protective effects may directly or indirectly combat many of the cardiotoxic effects associated with PCa, but continued research is needed. In a previous study by Wang et al., exercise was found to protect against chemotherapy induced cardiotoxicity as demonstrated by preventing the decrease in ejection fraction and fractional shortening seen in the group receiving doxorubicin and no concurrent exercise [7]. Unfortunately, limited research has been done to evaluate the protective effects of exercise in relation to PCa.

For those who are unable to exercise or those seeking a way to enhance the physiological benefits of exercise, dietary supplements containing botanicals may be of use. Phellodendron amurense (PhAm) bark extract has been found to have anti-proliferative effects on PCa by simultaneously targeting multiple unregulated pathways through which tumor cells grow [8]. Additionally, PhAm works to modulate inflammatory immune response in different cancer models that have also been mechanistically linked to cardiotoxic processes [9,10]. For this reason, we hypothesize that it may work as an exercise mimetic. Previous work by our group has found that PhAm similarly augments tumor aggressiveness in a PCa animal model in the same manner as exercise [10]. Current transcriptomic work by our group has suggests an antioxidant effect of PhAm that provides these benefits (unpublished, in review). We theorize that the protective capabilities of PhAm can be translated to cardioprotection.

While many studies have evaluated the effects of a simultaneous course of exercise and chemotherapy on the heart, few have investigated ways to fortify the heart prior to treatment to withstand its long term cardiotoxic effects, especially in the setting of PCa. Our study looks at the comparative and additive effects of exercise and PhAm as potential prehabilitation interventions to promote cardiovascular health in the presence of treatment naïve PCa.

METHODS

Animals.

Twenty-four, 10-week old, transgenic adenocarcinoma of the mouse prostate (TRAMP; stock #008215) mice were purchased from Jackson Labs (Bar Harbor, ME, USA). After a one-week acclimation period, the mice were randomly selected from the housing cage and assigned to one of four groups: control, exercise (Ex), PhAm or combo (ExPhAm; concurrent exercise and PhAm intervention) detailed below (Figure 1). Throughout the experiment, the mice were placed in a climate-controlled environment with a 12-hour light-dark cycle. They were also provided with food and water as needed. At necropsy, the mice were examined for gross organ anomalies. Animal care and handling were conducted in accordance with established humane guidelines and protocols, as approved by the University of Texas Health Science Center at San Antonio’s Institutional Animal Care and Use Committee.

Figure 1.

Study design

Voluntary wheel running.

Mice that were randomly assigned to the exercise group were given continual access to a running wheel with a diameter of 11.5 cm. The wheel rotations were monitored continually using a magnetic sensor (Med Associates, Inc., St. Albans, VT, USA). This approach of voluntary wheel running was chosen over forced wheel running (i.e. forced treadmill running), as it more closely emulates natural mouse behavior. This modality is consistent with our previous work that has demonstrated that wheel running activity can decrease the presence of aggressive tumor phenotypes and preserve muscle mass in TRAMP mice [10]. The use of a running wheel reflects activity consistent with the capabilities of normally active PCa patients and is inversely associated with mortality.

Preparation of PhAm diet.

PhAm power was pelleted into standard AIM-93G chow at a dose of 600 mg/kg based on positive results from previously published data by Dyets, Inc (Bethlehem, PA, USA). This diet consisted of cornstarch (397.5 g/kg), casein (200 g/kg), dextrose (132 g/kg), sucrose (100 g/kg), soybean oil (70 g/kg), cellulose (50 g/kg), salt mix #210050 (35 g/kg), vitamin mix #310025 (10 g/kg), L-cystine (3 g/kg), choline bitartrate (2.5 g/kg), and t-butylhydroquinone (0.014 g/kg). The manufacturer performed quality assurance and irradiation. The stability of PhAm present in the pellets was evaluated and reported previously [9,11,12]. Unrestricted access to the diet was provided and food intake was evaluated on a weekly basis. Similar monitoring was done for the group provided the standard diet.

Echocardiography.

Echocardiography was performed at baseline and end of study by a blinded technician. Baseline measurements (PRE) were done 24 hours prior to the start of the experimental period for each mouse. End of study (Post) measurements were done 24 hours after the completion of the experimental period and 24 hours prior to euthanasia. All system settings and parameters used for the echocardiography were maintained for each mouse at the respective timepoints.

Echocardiography was done using the Vevo 2100 system (Visualsonics, Inc., Toronto, Canada) with a MS400 linear array transducer (30 MHz). While under anesthesia (2% isoflurane and 1ml/min O2), mice were taped to imaging board. Chest hair was removed by depilatory cream (Nair, Johnson & Johnson, New Brunswick, NH) before any measurements. Electrode gel will be administered on chest. Electrocardiographic probe will be positioned directly above the heart and images will be acquired. Body temperature will be monitored and maintained between 34–37°C. The frame rates for M-mode were set to >200 frames per minute. M-mode short-axis images were captured at the level of the papillary muscles and the LV was bisected to obtain the ideal M-Mode selection. A minimum of 3 M-mode images were captured per mouse per timepoint. These images were then saved to a local hard drive and evaluated by a technician who was blinded to the experimental group. Traditional echocardiographic measurements of the left ventricle (LV) including ejection fraction (EF), fractional shortening (FS), cardiac output (CO), stroke volume (SV), end-diastolic dimension (EDD), end-systolic volume (ESV), end diastolic volume (EDV), global longitudinal strain (GLS), intraventricular thickness, and mass were taken. Representative image collected for the cardiac measures collected during systole and diastole are presented in Figure 2.

Figure 2.

Representative images of echocardiography procedure

Statistical analysis.

Inferential and descriptive statistics was performed on SPSS (IBM, Armonk, NY, USA) and visualized using GraphPad Prism 6 software (Boston, MA, USA). Between group and within group comparisons were assessed using a one-way and two-way analysis of variance (ANOVA). All animals were included in the analysis with the exception on one animal in the exercise group who was found dead midway through the study. Necropsy and pathology determined this animal had died due to idiopathic reason. Statistical significance was defined as p < 0.05 for all analyses and data are reported at mean ± standard deviation.

RESULTS

Body Mass.

No significant differences were observed between groups at baseline or end of study for body mass. There was a tendency (F(3,19)=2.640; p=0.08) at the end of the study for the ExPham group to have lower body mass compared to the control group (p=0.052). Within group differences demonstrated significantly higher body mass in each of the groups, with the exception of the exercise only group. Tumor mass and tumor free body mass measured at the end of study was not significant (Table 1).

Table 1.

Anthropometic data

	Body Mass
	Pre	Post	GU Mass	GU Free Body Mass*
Control	22.5 ± 4.1	28.38 ± 1.8	0.62 ± 0.1	27.9 ± 1.9
Exercise	25.1 ± 2.8	26.90 ± 1.1	0.60 ± 0.08	26.5 ± 1.1
PhAm	23.1 ± 1.8	27.25 ± 1.3	1.8 ± 2.2	25.0 ± 1.9
ExPhAm	23.9 ± 1.9	25.75 ± 1.8	0.7 ± 0.24	25.5 ± 2.8

GU - genitourinary;

^*GU Free Body Mass is representative of the body mass collected at the time of euthanasia substracting the mass of the GU complex that was collected to measure tumor mass

Physical Activity.

No significant differences were observed between the exercise (10.40 ± 1.8 km) and the ExPhAm (10.84 ± 2.0 km) group on average daily distance ran through the 12-week intervention period. Figure 3 presents group and individual variability in physical activity.

Figure 3.

Voluntary wheel running outcomes

Echocardiography outcomes.

Echocardiography was performed at baseline and after the 12-week intervention to assess cardiac function. No significant differences were observed between groups at baseline, however, a number of variations that were found to be significant at the end of the study (Table 2). In particular, significant differences were for cardiac output (F(3,19)=3.087; p=0.05), GLS (F(3,19)=3.251; p=0.045) and ejection fraction (F(3,19)=3.418; p=0.038). A tendency was observed for differences between group in stroke volume (F3,19)=2.627; p=0.08). Post hoc analysis revealed that the PhAm group was found to have higher stroke volume (p=0.01) and greater ejection fraction (p=0.026) compared to the ExPhAm group. PhAm group also presented with higher cardiac output when compared to the exercise only group (p=0.048). Finally, PhAm had a significantly lower GLS% when compared to the control group (p=0.05). No other significant differences were observed. Representative images are presented in Figure 4.

Table 2.

Echocardiographic measures

	Control		Exercise		PhAm		ExPhAm
	B	EOS	B	EOS	B	EOS	B	EOS
EDV(ul)	52.33 ± 7.6	45.18 ± 5.84	53.43 ± 7.4	44.9 ± 3.99	56.28 ± 15.0	49.92 ± 12.97	49.8 ± 6.0	46.5 ± 6.14
ESV(ul)	23.25 ± 5.1	17.7 ± 3.95	22.73 ± 5.1	17.78 ± 2.79	24.23 ± 10.4	16.82 ± 6.25	20.23 ± 3.7	19.83 ± 4.69
SV(ul)	29.08 ± 4.2	27.48 ± 2.88	30.73 ± 3.6	27.14 ± 1.28	32.00 ± 6.7	33.07 ± 7.46 a	29.55 ± 3.5	26.68 ± 3.44
FS (%)	31.43 ± 5.0	37.57 ± 3.25	30.70 ± 7.7	33.88 ± 5.6	32.6 ± 6.7	40.02 ± 8.37	31.4 0± 4.0	30.02 ± 10.94
CO (ml/min)	15.05 ± 2.5	14.58 ± 1.37	14.85 ± 1.9	13.16 ± 0.89	16.82 ± 2.7	17.3 ± 4.1 b	15.15± 1.18	14.1 ± 1.71
GLS (%)	−18.26 ± 3.1	−17.42 ± 2.89	−19.56 ± 1.9	−18.1 ± 3.63	−20.68 ± 2.5	−22.73 ± 3.62 c	−20.75 ± 2.3	−18.34 ±2.93
EF (%)	55.67 ± 5.7	60.83 ± 4.4	57.83 ± 5.2	60.4 ± 2.88	57.83 ± 8.1	67 ± 6.42 a	59.00 ± 4.3	57.33 ± 6.47
EDLVM	57.33 ± 5.8	69 ± 17.08	83.17 ± 17.9	61.4 ± 12.05	61.33 ± 10.5	61.67 ± 12.14	66.17 ± 7.9	59.17 ± 4.07
ESLVM	54.83 ± 7.1	62.17 ± 13.93	81.17 ± 17.4	57.6 ± 12.26	61.00 ± 11.2	59.33 ± 13.43	65.00 ± 8.7	53.83 ± 6.71
Heart Rate (bpm)	517.7 ± 44.00	531.73 ± 25.43	483.4 ± 32.9	486.08 ± 24.37	530.42 ± 33.9	523.88 ± 47.4	515.88 ± 29.2	527.82 ± 8.39
LV Mass (mg)	63.09 ± 3.52	72.56 ± 4.98	62.97 ± 7.0	69.03 ± 5.45	56.42 ± 11.7	64 ± 12.47	58.70 ± 5.8	63.73 ± 9.41
LV/BM Index (%)	2.88 ± 0.5	2.54 ± 0.26	2.40 ± 0.2	2.49 ± 0.14	2.45 ± 0.5	2.28 ± 0.39	2.44 ±0.1	2.46 ± 0.31

Data are mean ± standard deviatoin. B, Baseline; EOS, End of Study; PhAm, phellodendrun amurense; ExPhAm, exercise with phellodendrun amurense;

^ap≤0.01 compared to ExPhAm,

^bp<0.05 compared to exercise,

^cp<0.05 compared to control.

Figure 4.

Representative images of echocardiography outcomes

Within group differences were also observed. Left ventricular mass was found to be significantly higher (F(1, 19) = 17.23; p<0.001) at the end of the study for the control (p=0.001) and the PhAm (p=0.03) groups, respectively. When corrected with body mass, the control group had a significant reduction in the ratio (p=0.03). Though not significant, we observed that in the exercise and ExPhAm group the ratios increased with age, while the PhAm and control groups decreased substantially.

Thickness of the intraventricular septum during diastole (IVS-d) was also found to be significantly higher (F(1,19)=19.81; p<0.001) in the control (p=0.01) and PhAm group (p=0.004). Stroke volume (p=0.03) and cardiac output (p=0.02) were found to be significantly lower in the exercise group at the end of the 12-week exercise program.

An alternative method of evaluating the effect of our interventions was to analyze changes in our outcomes from baseline. This data is presented in Table 3. One-way ANOVA found significant differences in change from baseline for end systolic volume (F(3,19)=4.417; p=0.016), ejection fraction (F(3,19)=5.439; p=0.007), end systolic left ventricular mass (F(3,19)=7.945; p=0.001) and end diastolic left ventricular mass (F(3,13)=11.80; p<0.001). ExPhAm was found to have the smallest change in end systolic volume with significant differences observed compared to the PhAm (p=0.014) and a tendency for change compared to the control groups(p=0.055), respectively. PhAm was observed to have the highest change in ejection fraction while exercise was found to have a reduction in ejection fraction, leading to a significant difference between the two group (p=0.005). Exercise and ExPhAm were found to have greatest loss in end diastolic left ventricular mass compared to the other groups (p<0.05). Control mice had an increase in end systolic left ventricular mass compared to the other groups (p<0.05).

Table 3.

Echocardiography change from baseline

	Control	Exercise	PhAm	ExPhAm
EDV (ul)	−13.34 ± 5.75	−13.62 ± 10.52	−10.52 ± 14.4	−6.37 ± 8.22
ESV (ul)	−23.7 ± 4.86	−17.43 ± 11.47	−28.93 ± 18.25	−1.96 ± 15.57 a b
SV (ul)	−4.36 ± 13.53	−10.11 ± 11.89	3.3 ± 10.96	−9.55 ± 7.89
FS (%)	21.83 ± 21.37	13.05 ± 29.33	23.87 ± 16.99	−4.23 ± 34.35
CO (ml/min)	−1.06 ± 17.82	−11.98 ± 7.65	2.22 ± 13.01	−7.1 ± 6.63
GLS (%)	−1.85 ± 24.15	−6.38 ± 21.17	9.62 ± 7.28	−10.72 ± 18.03
EF (%)	9.83 ± 9.29	3.84 ± 5.93	16.9 ± 11.46	−2.91 ± 7.15 c
EDLVM	20.28 ± 25.49	−29.21 ± 14.61 d e	0.89 ± 13.59	−9.67 ± 10.83 f
ESLVM	12.87 ± 14.81	−31.81 ± 14.16 g h	−2.7 ± 13.59	−16.69 ± 9.27 g
Heart Rate (bpm)	3.39 ± 10.82	−1.08 ± 7.65	−1.06 ± 9.16	2.56 ± 5.50
LV Mass (mg)	15.01 ± 4.92	10.72 ± 15.21	15.24 ± 18.71	8.74 ± 13.34
LV/BM Index (%)	−10.33 ± 11.47	4.49 ± 11.04	−4.18 ± 19.05	1.14 ± 15.24

Data are mean ± standard deviatoin and were compared with a one-way ANOVA. PhAm, phellodendrun amurense; ExPhAm, exercise with phellodendrun amurense;

^ap=0.055 compared to control,

^bp=0.014 compared to PhAm,

^cp=0.005 compared to PhAm;

^dp=0.04 compared to PhAm;

^ep<0.001 compared to control

^fp=0.033 compared to control

^gp<0.01 compared to control

^hp=0.008 compalred to PhAm

DISCUSSION

Briefly, the purpose of this study was to investigate the individual and additive effect of exercise and PhAm on outcomes of cardiac function as measured by echocardiography in a treatment naïve PCa mouse model. This study found that exercise is the driving factor in preserving cardiac function as represented in changes from baseline, either in the exercise only group or when combined in the ExPhAm group.

Few studies have examined the relationship between PCa and cardiac function, and these tend to show no significant changes in EDV, ESV, SV, FS, and EF [13]. However, a significant decrease in LV mass [13] is typically observed, indicating atrophy [14]. This reflects what was observed in the control group of this study and is seen with the decrease in IVS-d [15]. The exercise intervention prevented an increase in LV wall thickness and decrease in LV diameter, thus preventing significant hypertrophy.

Similarly, aging is known to worsen overall cardiac function [16]. Previous studies have shown that aging is negatively associated [17] with LV/Body Weight index. Exercise has been shown to prevent phenotypic aging and promote a younger phenotype in mice with PCa. This can be seen through an increase in LV/ Body Weight index [17]. In this study, mice in both the Ex and ExPhAm groups showed clinically relevant increases in this index, indicating that they resisted typical aging-related cardiovascular changes that were observed in the groups that did not take part in exercise. Additionally, aging in mice is associated with increases in EDV, ESV, BW, and LV mass [17]. Aging is associated with decreases in SV, FS, CO, and EF [18]. These effects can be noted through examination of the control group and can be prevented with voluntary exercise.

Effect of Exercise and cardiac function

In general, aerobic exercise has been proven to enhance cardiac function. Exercise interventions done specifically in mice tend to show decreased IVS-d thickness thus preventing significant LV hypertrophy of the heart walls [17]. Although relative wall thickness decreases after an exercise intervention, LV mass increases [17], indicating hypertrophy of the heart. This is theorized to be due to fortification of the heart, rather than due to pathological hypertrophy seen in conditions such as hypertension [4]. For this reason, relative wall thickness is a better measure to distinguish between beneficial and harmful causes of LV hypertrophy, specifically in the context of exercise. Exercise is also typically associated with increases in LV/ BW index [17] and LVID [19]. This is mostly consistent with the data in this study, as the Ex and ExPhAm groups prevented significant increases relative wall thickness. However, the results show no significant changes in LV mass, indicating no significant exercise-induced remodeling of the heart. The overall exercise effect also increased the LV/ BW index, preventing declining cardiac function associated with aging as expected [17].

Research shows that exercise promotes physiological adaptations which increase cardiac output [20]. However, this study found a significant decrease in cardiac output after the exercise intervention. We also found a significant decrease in SV, despite no significant changes in EF or FS. Additionally, previous studies have found that heart rate will decrease [21] after an exercise intervention, while this study found no significant differences. Since SV and HR are variables needed to calculate CO, and CO is predicted to increase [4] and HR is predicted to decrease after an exercise intervention, our results should have shown a significant increase in SV, which was not seen. This is an interesting finding and one that warrants further investigation. Overall, the outcomes of this study support our hypothesis that exercise enhances cardiac function.

Effect of PhAm

PhAm was studied as a potential nonpharmacologic intervention and alternative to exercise, particularly in those who are unable to exercise due to comorbidities. We hypothesized that PhAm could mimic the cardiac benefits of exercise due to its anti-inflammatory properties that can be cardioprotective against the negative effects of PCa and aging [8–10].

Contrary to our expectations, the PhAm group showed similar degree of hypertrophy and aging to the control group. The PhAm group showed a significant increase in LV mass, and IVS-d thickness. This group also showed a decrease in LV/BW index, indicating that PhAm alone was not protective against the effects of aging [17]. Hence, our initial hypothesis that PhAm may act as a cardioprotective agent was not supported by the data, as it did not prevent against hypertrophy or the cardiac effects of aging.

Limitations to this study

This study has several limitations that should be addressed. The total sample size of the study was small, with only 24 total mice among all 4 groups. Next, this study did not include a non-tumor comparison group. This method was chosen to highlight changes specifically relating to exercise and PhAm. However, examining the effect of exercise and PhAm on tumor and non-tumor groups would be of interest for future studies. Lastly, this study does not include mechanistic data showing physiological changes. Specific tumor markers and blood enzymes were not measures and would be useful to better understand the mechanism through which these groups alter cardiac function. Future studies will aim to measure serum levels of tumor and inflammatory markers in each intervention to better understand the mechanisms through which each treatment works. This would enhance our understanding of how PhAm works in the context of PCa, aging, and exercise. Additionally, a similar study with a larger sample size should be conducted to prevent a confounding affect due to chance.

Conclusion

Overall, the outcomes of this study suggest that exercise is effective in preventing significant left ventricular hypertrophy when evaluated using relative wall thickness. Although we predicted PhAm to have similar effects as the exercise interventions, we found no such relationship in this study.

Exercise was also the driving force in the ExPhAm group. These findings suggest that prehabilitative exercise may resist the cardiotoxic effects associated with PCa and aging.

Abbreviations

IVS

Intraventricular septum

LVID

left ventricular internal dimension

EF

ejection fraction

FS

fractional shortening

LV Mass

left ventricular mass

LV Vol

left ventricular volume

LV:BM

left ventricular mass to body mass ratio

d

diastole

s

systole

EDV

End diastolic volume

ESV

End systolic volume

SV

Stroke Volume

EDLVM

End diastolic left ventricular mass

ESLVM

End systolic left ventricular mass

CO

Cardiac output

GLS

Global longitudinal strain

Data Availability:

Source data for this study are openly available at 10.6084/m9.figshare.28908380