An Evaluation of Cardiac Health in the Spontaneously Hypertensive Rat Colony: Implications of Evolutionary Driven Increases in Concentric Hypertrophy

Abstract

Background

The Spontaneously Hypertensive Rat (SHR) Colony was established in 1963 and is the most commonly used rodent model for studying heart failure (HF). Ideally, animal models should recapitulate the clinical disease as closely as possible. Any drift in a genetic model may create a new model that no longer adequately represents the human pathology. Further, instability overtime may lead to conflicting data between laboratories and/or irreproducible results. While systolic blood pressure (SBP) is closely monitored during inbreeding, the sequelae of HF (e.g., cardiac hypertrophy) are not. Thus, the object of this review was to investigate whether the hypertension-induced sequelae of HF in the SHR have remained stable after decades of inbreeding.

Methods

A systematic review was performed to evaluate indices of cardiovascular health in the SHR over the past 60 years. For post hoc statistical analyses, studies were separated into 2 cohorts: Initial (mid to late 1900s) and Current (early 2000s to present) Colony SHRs. Wistar-Kyoto rats (WKY) were used as controls.

Results

SBP was consistent between Initial and Current Colony SHRs. However, Current Colony SHRs presented with increased concentric hypertrophy (i.e., elevated heart weight and posterior wall thickness) while cardiac output remained consistent. Since these changes were not observed in the WKY controls, cardiac-derived changes in Current Colony SHRs were unlikely due to differences in environmental conditions.

Conclusions

Together, these data firmly establish a cardiac-based phenotypic shift in the SHR model and provide important insights into the beneficial function of concentric hypertrophy in hypertension-induced HF.

Graphical Abstract

Heart failure (HF) is a global health concern that affects nearly 26 million people.¹ Despite decades of HF research, the translatability of animal studies into clinical practice remains a significant barrier. As a result, careful consideration must be taken when selecting animal models to ensure they are predictable in their pathogenesis and representative of human pathophysiology.² The Spontaneously Hypertensive Rat (SHR) is the most commonly used experimental model to study HF (Figure 1a).³ Despite the widespread use of this model, whether the hypertension-induced sequelae of HF (e.g., cardiac hypertrophy and dysfunction) is maintained overtime has yet to be evaluated.

Figure 1.

The SHR is the most commonly used hypertensive model. (a) Prevalence of the SHR (genetic) and transverse aortic constriction (surgical) models of hypertension were assessed using a PubMed key-term search. (b) Schematic illustrating the establishment of the SHR Colony and its diverse research uses. Abbreviation: SHR, Spontaneously Hypertensive Rat. Created with BioRender.com.

The SHR Colony was established in 1963 by Kyoto University investigators, and is used to study various conditions such as HF, attention deficit and hyperactivity disorder, diabetes, and obesity (Figure 1b).^3–6 To ensure the hypertensive phenotype is retained in progeny, systolic blood pressure (SBP) of potential breeders is closely monitored. However, the sequelae of hypertension, which lead to HF are not monitored to the same degree. As a result, we postulate that over the past 60 years, the severity of HF in the SHRs has diminished.

Cardiovascular fitness is an important component of reproductive health. Improvements in cardiovascular health are associated with increased breeding rates and more successful pregnancies.^7,8 Previous findings demonstrate that natural selection can occur in a number of animal models, resulting in a beneficial shifting of the animals’ original phenotype.^9–11 Unlike humans, the SHRs develop hypertension at their reproductive age (i.e., when animals are selected for breeding), providing the opportunity for natural selection toward adaptive traits for hypertension-induced HF. Therefore, the purpose of the present study was to determine whether SHRs have evolved over time to the cardiac sequelae of hypertension. Given that cardiovascular health is impaired in the SHRs at the time of breeding, we hypothesized that over the past 60 years the SHRs have adapted to better tolerate their chronic hypertension.

METHODS

Systematic review

In this perspective, we used a systematic review followed by a post hoc analysis to support the notion that, through natural selection, the SHR model has drifted. To evaluate cardiovascular health in the SHR Colony over the past 60 years, a retrospective systematic review of the literature was performed to identify articles that utilized SHRs in their study. Articles were identified using a key-term search in the PubMed database (June–July 2020). First, to determine which model of hypertension was the most prevalent within the literature (i.e., SHR—genetic model or TAC—surgical model) the key-terms “SHR” and “transverse aortic constriction” were searched on the PubMed database. For each term, the number of publications per year, from 1963 to 2020, was plotted in a line graph to comparatively assess the prevalence of these 2 models. Second, to fully characterize the cardiac health of the SHR Colony, articles were acquired based on the following 3 methodologies of interest: morphometry, echocardiography, and invasive hemodynamics.

The morphometry key-term search was used to determine whether the SHRs underwent cardiac structural changes over time. The following key-terms were used: (SHR and heart weight), (SHR and left ventr* mass), (SHR and left ventr* weight), (SHR and right ventr* mass), (SHR and right ventr* weight). The asterisks were used to allow for truncation (i.e., retrieve studies that measured either ventricle or ventricular mass). The echocardiography key-term search was used as a secondary means of assessing cardiac structure in the SHRs over time, and additionally, to assess cardiac function. The following key-terms were used: (SHR and ECHO), (SHR and echocardiography). Additionally, for the echocardiography data when provided with sufficient parameters (e.g., LVESd and LVEDd) additional parameters were calculated (e.g., fractional shortening) using the VisualSonics Vevo 770 imaging system (VisualSonics, Toronto, ON, Canada) and included in the analysis. The invasive hemodynamic key-term search was used to investigate systolic and diastolic function in the SHR Colony. The following key-terms were used: (SHR and LVP), (SHR and LVSP), (SHR and LVEDP), and (SHR and dP/dt). A detailed summary of the methodology is outlined in Table 1.

Table 1:

Overview of study methodology

Category	Parameters of Interest	Initial Colony SHRs	Current Colony SHRs
Morphometry	HW, BW	1974 – 1985 (n = 34)	2016 – 2020 (n = 42)
Echocardiography	LVEDd, LVESd, PWTd, FS, EF, SV, CO, HR	1986 – 2006 (n = 19)	2017 – 2020 (n = 36)
Invasive Hemodynamics	dP/dtMax, dP/dtMin, LVSP, LVEDP	1981 – 1991 (n = 13)	2010 – 2020 (n = 20)

Abbreviations: HW, heart weight; BW, body weight; LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; PWTd, diastolic posterior wall thickness; FS, fractional shortening; EF, ejection fraction; SV, stroke volume; CO, cardiac output; HR, heart rate; dP/dt_Max, maximal rate of pressure change in the left ventricle; LVSP, left ventricle systolic pressure; dP/dt_Min, minimal rate of pressure change in the left ventricle, LVEDP; left ventricle end-diastolic pressure. Time frames for the Initial Colony SHRs and Current Colony SHRs differ for each category of interest (i.e., morphometry, echocardiography and invasive hemodynamics) due to reporting differences for each technique and to ensure enough time was left between the two cohorts to capture potential shifts in the model. n-values indicate the number of studies that met the inclusion criteria from the key-term search.

Inclusion and exclusion criteria

Papers were selected for statistical analysis if they were peer reviewed and available online (inclusion criteria). Additionally, each article must have explicitly stated that the SHRs were males and included the age of rats used. All papers that did not meet these criteria were immediately excluded. The remaining papers were analyzed more thoroughly by reading the text in full to appraise the quality of techniques used (e.g., must have used a mouse echocardiography probe and not a human neonatal echocardiography probe) and to ensure the reported data were adequately described (e.g., must have specified the type of heart weight collected: whole heart weight, ventricle weight, etc.).

Studies where substrains of the SHR Colony (e.g., stroke-prone/stroke-resistant SHR and lean/obese SHRs) were used, were excluded. This was done to minimize the amount of variables that could affect the interpretation of the results. Hemodynamic measures were only included if they were obtained via left ventricle catheterization. Studies that utilized langendorff or telemetry were excluded. This was done to ensure consistency between the Initial and Current Colony groups, as these techniques were not often used in the late 1900s. Lastly, data were only collected from studies where no extreme experimental conditions were imposed upon the background that would overtly alter the course of the pathophysiology (i.e., no concurrent myocardial infarction, nephrectomy, or pharmacological intervention were introduced to the SHRs prior to the collection of cardiac data).

To account for potential differences in methodology (i.e., environment, equipment, and investigator techniques), control data were collected and analyzed from Wistar-Kyoto (WKY) rats (i.e., rats used to establish the SHR line via selective breeding). Since WKY rats are often used as SHR controls, we obtained WKY datasets (to be used as the SHR comparative baseline) from studies which met our SHR inclusion criteria.

Ideally, the time frame of each methodology (i.e., morphometry, echocardiography, and invasive hemodynamics) would be consistent within each SHR Colony (i.e., Initial vs. Current Colony SHRs). However, given differences in technique prevalence and establishment, and the need to maintain a sufficient time period separation (i.e., >10 years) between the Initial and Current Colony, date ranges between the investigated techniques varied slightly. As a result of these accommodations, we were able to collect sufficient data points for analyses.

Statistical analysis

Post hoc statistical analyses were performed using Prism 8 (GraphPad Software). The Anderson–Darling normality test was used to determine whether each dataset was normally distributed. Statistical significance between Initial and Current Colony SHR or WKY rats was determined using a parametric t-test when the data were normally distributed and a nonparametric t-test when the data failed the normality test. The threshold for significance was P < 0.05 in all cases unless otherwise specified.

RESULTS

The severity of hypertension was consistent within the SHR Colony over time

Initially, hypertension causes compensatory cardiac remodeling which, in the long term, leads to the development of overt HF.¹² The SHR Colony was originally established with the purpose of creating a genetically stable model of hypertension. However, whether the severity of the SHR’s hypertension has remained stable over the past 60 years has not yet been assessed. To evaluate whether the SHRs’ hypertensive phenotype has been maintained, SBP was compared between Initial and Current Colony SHRs. In both colonies, SBP increased from 5 to 20 weeks of age and plateaued thereafter at 195 mm Hg (Figure 2). These data demonstrate that the severity of hypertension within the SHR Colony has remained consistent over time.

Figure 2.

Mean SBPs were obtained by tail-cuff plethysmography. Abbreviations: SBP, systolic blood pressure; SHR, Spontaneously Hypertensive Rat.

Current Colony SHRs had increased left ventricular concentric hypertrophy

Hypertension causes compensatory cardiac hypertrophy, which aims to normalize wall stress to that of a normotensive heart.³ The magnitude of the acute hypertrophic response is driven by the severity of hypertension.^13,14 To investigate whether the SHRs’ hypertrophic response has remained stable over the past 60 years, we evaluated heart weight in the Initial and Current Colony. Body weight (Figure 3a) was evaluated alongside heart weight (Figure 3b) to ensure changes in heart weight were not a product of fluctuations in body weight over time (in weeks). Adult rats (i.e., age >29 weeks) were used for statistical comparisons given that the hypertensive phenotype has stabilized and the SHRs have developed compensatory cardiac hypertrophy.^4,15 While body weights were unchanged (Figure 3c), Current Colony SHRs showed significantly increased heart weights (Figure 3d). Since heart and body weights remained consistent in the WKY control rats (Figure 3e–h), changes in the Current Colony SHRs are unlikely due to differences in environmental factors,¹⁶ such as animal housing and feed. Overall, these results indicate that, although the severity of hypertension has remained consistent within the SHRs over time, Current Colony SHRs undergo greater hypertrophic remodeling.

Figure 3.

Mean (a) body weights and (b) heart weights of the Initial and Current Colony SHRs over time. (c) Statistical analysis of mean body weights in adult (29 weeks+) SHRs. (d) Statistical analysis of mean heart weights in adult SHRs. Mean (e) body weights and (f) heart weights of the Initial and Current Colony WKYs over time. (g) Statistical analysis of mean body weights in adult WKYs. (h) Statistical analysis of mean heart weights in adult WKYs. Abbreviations: SHR, Spontaneously Hypertensive Rat; WKY, Wistar-Kyoto Rats. *P < 0.05.

To further investigate changes in cardiac structure and function within the Current Colony SHRs, echocardiographic data were assessed (Table 2). Current Colony SHRs showed an increase in diastolic posterior wall thickness, which is indicative of concentric hypertrophy in the left ventricle. Despite a decrease in mean stroke volume (SV), which typically accompanies concentric hypertrophy, mean cardiac output in the Current Colony SHRs was maintained. This response is likely a result of a compensatory increase in heart rate (HR), given that cardiac output is the product of SV × HR. To ensure the hypertrophic changes in the SHRs were not due to differences in external factors, echocardiographic parameters were also assessed in the Initial and Current Colony WKY control rats (Table 3). Both cardiac structure and function were consistent between all control animals. Collectively, these data further demonstrate that chronic hypertension in Current Colony SHRs resulted in a heightened hypertrophic response.

Table 2:

Echocardiographic parameters of Initial and Current Colony SHRs

Parameter	Initial SHRs	Current SHRs
LVEDd (mm)	7.414 ± 0.201	7.240 ± 0.220
LVESd (mm)	4.278 ± 0.225	4.385 ± 0.228
PWTd (mm)	1.736 ± 0.095	2.108 ± 0.091*
FS (%)	42.59 ± 2.081	41.86 ± 1.352
EF (%)	73.29 ± 2.011	70.64 ± 1.879
SV (ml)	219.5 ± 12.14	180.9 ± 5.92*
CO (ml/min)	61.50 ± 3.560	63.36 ± 3.144
HR (beats/min)	304.6 ± 14.58	352.8 ± 11.50*

Data are presented as means ± SEM . Abbreviations: LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; PWTd, diastolic posterior wall thickness; FS, fractional shortening; EF, ejection fraction; SV, stroke volume; CO, cardiac output; HR, heart rate. * = p < 0.05 comparison between Initial and Current Colony SHRs.

Table 3:

Echocardiographic parameters of Initial and Current Colony WKYs

Parameter	Initial WKYs	Current WKYs
LVEDd (mm)	7.350 ± 0.177	7.211 ± 0.208
LVESd (mm)	4.339 ± 0.127	4.074 ± 0.173
PWTd (mm)	1.364 ± 0.098	1.637 ± 0.090
FS (%)	41.41 ± 1.023	44.62 ± 1.332
EF (%)	73.43 ± 1.780	76.93 ± 1.910
SV (ml)	196.5 ± 11.95	200.4 ± 12.52
CO (ml/min)	67.15 ± 2.542	75.27 ± 9.390
HR (beats/min)	328.9 ± 11.14	334.5 ± 19.30

Data are presented as means ± SEM. No statistically significant differences were found in any of the parameters examined. Abbreviations: LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; PWTd, diastolic posterior wall thickness; FS, fractional shortening; EF, ejection fraction; SV, stroke volume; CO, cardiac output; HR, heart rate; WKY, Wistar-Kyoto Rats.

Cardiac contraction and relaxation were unchanged within the SHR Colony

Previous findings have established that increased cardiac hypertrophy results in greater left ventricular wall stiffness, which leads to elevated diastolic filling pressures and diastolic dysfunction.¹⁷ While echocardiography is often used as a noninvasive measure of cardiac function, invasive hemodynamic assessment is the gold standard.¹⁸ To understand the functional ramifications of cardiac remodeling in the Current Colony SHRs, several invasive hemodynamic parameters were assessed (Figure 4). Overall, no significant differences were observed in indices of cardiac contraction (i.e., dP/dt_Max) and relaxation (i.e., dP/dt_Min) within either the SHR or WKY Colonies. However, several atypical trends in these datasets were observed. In both the SHR and WKY Colonies, there was a large degree of variance in the rate parameters (Supplementary Tables 1 and 2). For instance, in Initial Colony SHRs, dP/dt_Max had a mean value of 9,356 mmHg/second, a standard deviation of 4,942 mmHg/second, with individual data points spanning a range of 13,279 mmHg/second. Notably, left ventricle end-diastolic pressure (LVEDP), an index of diastolic function, significantly increased in both the Current Colony SHR and WKY rats. Although this finding was expected in the SHRs, given that both chronic hypertension and concentric hypertrophy lead to elevations in LVEDP,¹⁹ a similar 2-fold increase in normotensive WKY control rats was unexpected. Data analyses were further complicated by the incomplete reporting of left ventricular systolic pressures (LVSPs). This limited our ability to conduct a comprehensive hemodynamic comparison between the Initial and Current Colony SHRs. As a result, our findings indicate that, while the LVEDP data were inconclusive (as LVEDP changed similarly in SHR and WKY control animals), cardiac contraction and relaxation remained unchanged within the SHR Colony.

Figure 4.

Mean values of Initial and Current Colony SHRs: (a) dP/dt_Max, (b) dP/dt_Min, and (c) LVEDP over time. Mean values of Initial and Current Colony WKYs: (d) dP/dt_Max, (e) dP/dt_Min, and (f) LVEDP over time. *P < 0.05. Abbreviations: dP/dt_Max, maximal rate of pressure change in the left ventricle; dP/dt_Min, minimal rate of pressure change in the left ventricle; LVEDP, left ventricle end-diastolic pressure; LVSP, left ventricle systolic pressure; SHR, Spontaneously Hypertensive Rat; WKY, Wistar-Kyoto Rats. *P < 0.05.

DISCUSSION

This study is the first to report a cardiac-based phenotypic change in the SHR Colony since its initial establishment in 1963. In this review, we examined several indices of cardiac structure and function in the SHR Colony over the past 60 years. Given that the hypertensive phenotype is present at the time of breeding and will impact cardiovascular fitness, there is opportunity for natural selection toward positive adaptive traits. Here, we show that while the severity of hypertensive remained stable in the SHR Colony, the cardiac pathophysiology sequelae of hypertension has changed over time toward greater concentric hypertrophy (i.e., increased heart weight and diastolic posterior wall thickness). Concentric hypertrophy occurs in response to resistance exercise without pathology. Conversely, concentric hypertrophy is a universal response to systemic hypertension and widely recognized as a risk factor for increased mortality; pharmacological inhibition of hypertrophy generally leads to improved left ventricle dysfunction. This work establishes that concentric hypertrophy is beneficial in a hypertension setting when developed through natural selection. Together, these data firmly establish a phenotypic shift in the SHR model and provide important insights into the adaptive hypertrophic response to hypertension-induced HF.

The SHR Colony was originally developed as a model of human essential hypertension.⁴ As a result, blood pressure is the only parameter considered when selecting breeders to maintain the colony. Typically, in response to chronic pressure stress (i.e., hypertension), the left ventricle will hypertrophy concentrically to minimize wall stress and maintain cardiac contractile forces.²⁰ However, in the long term, the accompanying increase in left ventricle wall stiffness will contribute to the progression of HF.²⁰ Interestingly, although the severity of hypertension had been preserved in the SHR Colony, Current Colony SHRs had increased concentric cardiac hypertrophy. Since the increase in hypertrophy was not driven by a more severe pressure stress, another factor (e.g., natural selection) was likely responsible for the shift in the colony.

Indeed, the phenotypic changes observed in the Current Colony SHRs were not caused by a change in pressure stress, however, these changes may be due to natural selection toward positive adaptive traits. Classifying a species’ phenotypic change as an adaptation, first requires identifying the functionality to which it serves.²¹ In regards to the Current Colony SHRs, the increase in concentric hypertrophy functioned to minimize wall stress and maintain cardiac function. Additionally, in order for a phenotypic trait to be selected for over time, the adaptation must increase the survivability and reproductive fitness of the individual.²² In Current Colony SHRs, this adaptation would have increased reproductive fitness,^7,8 thus, SHRs with the greatest potential for reproduction may have been selected. Furthermore, the SHR Colony is under a high selection intensity (i.e., frequent breeding, and many progeny descending from 1 breeding pair). This further increases the likelihood that natural selection is the causative mechanism behind this change.

Although increases in concentric hypertrophy can be driven by either evolution (e.g., SHR) or pathology (e.g., myocardial infarction), their effect on cardiac function differs. On the one hand, pathological hypertrophy is initially beneficial, however, over time, it causes worsened cardiac function and exacerbates the progression of HF.²⁰ On the other hand, increased concentric hypertrophy in Current Colony SHRs had no effect on cardiac contraction or relaxation, indicating that cardiac hypertrophy, driven by natural selection, is divergent from hypertrophy caused by pathological stress. Ultimately, these findings provide novel insights on the function of natural selection driven increases in concentric hypertrophy in the progression of hypertension-induced HF.

The left ventricle hemodynamic response to chronic pressure overload is well established.¹³ Elevated systemic blood pressure leads to an increase in LVSP to maintain cardiac output. This increase in force production requires the heart to undergo concentric remodeling, adding sarcomeres in parallel to normalize the increase in systolic wall stress.¹⁹ Additionally, chronic hypertension is associated with diastolic dysfunction, characterized by elevated end-diastolic filling pressures for a given end-diastolic volume.²³ Interestingly, although the severity of hypertension was not different between SHR colonies, the Current Colony SHRs showed elevated LVEDP, which was concomitant with an increase in concentric hypertrophy compared with Initial Colony SHRs. This increase in diastolic filling pressure may be attributed to a decrease in ventricular compliance—a result of more severe left ventricle hypertrophy in Current Colony SHRs.^13,24,25 However, we also observed an increase in LVEDP in the Current WKY Colony compared with the Initial Colony in the absence of hypertrophic remodeling. Therefore, it is possible that these changes are a result of altered ventricular loading conditions, genetic modifications that decreased compliance, or external factors (i.e., quality of equipment, individual investigator technique, and anesthetic type) that are not reflective of a true increase in myocardial stiffness.

Parameters of cardiac contraction and relaxation were also evaluated in the SHR and WKY Colonies to further characterize cardiac function. No differences were found in either colony, which may be due to large interstudy variability in each of the parameters measured, limiting our ability to detect group differences. We observed large SDs for dP/dt_Max and dP/dt_Min, and data values, which are atypical of healthy animals or animals with severe HF. As large variability was observed in both SHR and WKY colonies, this suggests that hemodynamic data may be influenced by external factors (e.g., investigator technique, equipment, software, signal artifacts, and anesthetic type/depth), rather than being reflective of a true change in cardiac physiology. Indeed, previous literature has reported a lack of standardization in the collection and reporting of cardiac data.^24,26 Our findings support the need for greater attention to equipment calibration, control of environmental factors (e.g., body temperature and anesthetic type/depth), and improved diligence in reporting hemodynamic methods, including commercial sources of equipment, software analysis programs, and sampling rates. This will improve study reproducibility and accuracy of future comparative analyses of published data.

In summary, while pressure stress (i.e., SBP) remained consistent within the SHR Colony, several indices of cardiac structure and function changed over time. These findings establish that natural selection increased the concentric hypertrophic response to hypertension, identifying this attribute as a positive adaptive trait. Together, this study identifies the presence of a hypertrophic adaptation in response to a chronic pressure stress in SHRs, firmly establishing a phenotypic shift in the SHR Colony since its initial establishment in 1963.

Limitations

This study has limitations. (i) For all datasets each point depicted on the graphs represent the mean value from a previously published study. As a result, there is potential for variation between each data point based on the level of experience of the investigator, quality of equipment used, age of the rats, number of rats used per study, environmental conditions in which the animals were maintained and type of anesthesia used. However, this limitation was addressed by including the WKY control group as a baseline reference for all the parameters analyzed, which serves as a reference group for inter-/intrastudy variability. (ii) The study selection process was only conducted by 1 reviewer. Historically, it has been recommended to utilize 2 independent reviewers to select studies, however, a recent publication indicated that a 1-step selection process is more time efficient and does not sacrifice the quality of reporting or lead to incorrect/misleading results.²⁷ (iii) Only male SHR data were included in the statistical analysis for all parameters investigated. This was due to an insufficient amount of studies that utilized female SHRs and that female SHRs do not develop the same severity of hypertension, and overt signs of HF (i.e., ventricular stiffness and dilation) as age-matched males.²⁸ Ideally, we would have assessed if the hypertension-induced sequelae of HF had evolved in both male and female SHRs, given that we know in humans there are also sex-based differences in HF. However, this was not feasible based on the limitation to the existing literature and historical inequity for experimental design. A comparative analysis between male and female SHRs is required to fully understand the pathophysiological differences in HF development in this model and is an opportunity for future studies.

FUNDING

This study was supported by National Science and Engineering Research Council, Canadian Institutes of Health Research, and Heart and Stroke Foundation of Canada grants (to J.A. Simpson and K.R. Brunt).

DISCLOSURE

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.