Abstract
The minipig has been increasingly recognized as a valid alternative to canines and nonhuman primates in regulatory toxicity. This article presents the results of cardiovascular assessments in the Yucatan, Hanford, Sinclair, and Göttingen minipigs conducted during nonclinical investigations and control toxicity testing. Cardiac electrophysiology was obtained using clinical electrocardiogram and surgical monitor units. Peripheral vessel diameter, velocity, and flow were obtained by Doppler ultrasonography, and cardiac vessel diameter was obtained postmortem. Anatomic parameters were obtained at necropsy. Histopathology assessments were conducted on heart, blood vessels, and kidneys. Collected data were compared to published cardiovascular measurements of adult humans to illustrate similarities and differences. Each lineage of minipigs was found to have specific anatomic and physiologic characteristics that may accurately reflect response of human cardiovascular systems in clinical investigations and toxicity testing. In conclusion, the interspecies similarities between the cardiovascular systems make these lineages of minipigs suitable as models for the human counterpart. In addition, these reported differences between lineages will aid investigators in selecting a relevant lineage of minipigs if specific cardiovascular parameters are required during drug safety evaluation.
Introduction
Miniature swine, or minipigs or miniswine, have been increasingly recognized as a suitable nonrodent model in place of canines and nonhuman primates in drug safety studies. 1,2 They are also being used more frequently for cardiovascular safety pharmacology studies, since their anatomic and physiologic similarities to humans favor their use. 1 Although several studies have been conducted to determine various cardiovascular characteristics of swine, including comparing regular pig hearts to human hearts, 3 comparing electrocardiograms (ECGs) of standard pigs to minipigs, 4 and creating human disease models, 4,5 the objective of this publication is specifically to create a compilation of normal and background cardiovascular information of multiple breeds of minipigs. The results of cardiovascular assessments in different breeds of physically normal, healthy minipigs conducted during clinical investigations and control toxicity testing are presented. This information will enable investigators to appropriately select the breed that exhibits cardiovascular parameters required for individual drug safety evaluations and also to become familiar with normal findings for the minipig in order to adequately interpret study findings.
Materials and methods
Information was collected from control animals in various toxicity studies and clinical investigations performed at Sinclair Research Center, LLC (SRC) from 2005 through 2014. Certain Göttingen information, specifically heart weight (HW), body weight (BW), 6 and background histopathology, 7 was gathered from previously published sources and incorporated into the information for completeness and comparison purposes.
Animals
Sinclair, Hanford, and Yucatan minipigs were obtained from Sinclair BioResources, LLC. Göttingen minipigs were purchased from Marshall BioResources (Clyde, NY). Anywhere from 5 to 119 animals of each breed were used to collect each parameter. All procedures and animal care conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the SRC Animal Care and Use Committee.
Housing
All minipigs were individually housed, with the exception of four females that were group-housed two to an enclosure. All pens were stainless steel with a minimum of 3 × 5.5 ft floor space. Pen walls were either completely solid or had solid lower walls with upper vertical bars; all front gates were made of vertical bars. Flooring was made of raised polyvinyl chloride-coated self-spanned metal. A metal chain was suspended in each pen for enrichment. Harlan® Teklad #7037C (Madison, WI), Purina® Sinclair S9 (Montgomery City, MO), or other equivalent miniswine diet was fed at maintenance once a day. Deep well water was provided ad libitum. Ambient temperature was maintained at 15°C–30°C (59°F–86°F). Humidity ranged from 12.8% to 100%. Lighting was maintained at a 12-h light/dark cycle and lights were turned on at 6:00 a.m.
Cardiovascular parameters
HW and kidney weights (KW) were recorded at SRC. Göttingen HW and BW were obtained from Bollen et al. 6 and incorporated for comparison purposes.
External iliac and femoral artery measurements were assessed in vivo using Siemens G60 ultrasound (Washington, DC) and OEC Medical C-arm fluoroscope (Salt Lake City, UT) series 9600. Left anterior descending artery internal diameter (LAD ID), left circumflex artery internal diameter (LCX ID), right coronary ID, aorta outer diameter (OD) and ID, and carotid ID were measured postmortem with electronic calipers. Heart height and circumference were also measured postmortem.
ECGs were obtained from 3- to 9-month-old minipigs using Schiller AT-2 or AT-2 plus multichannel ECG (Baar, CH) units. Animals were placed in sternal recumbency in a sling or in lateral recumbency on a nonconducting surface. Hair was clipped prior to attaching electrodes. Leads I, II, and III were the minimum recording requirement; leads aVR, aVL, and aVF were generally included as well. ECGs were recorded at a paper speed of 25 mm/s for 20–60 s and then briefly recorded at 50 mm/s. Results were read by a veterinary cardiologist, and the electrographic parameters heart rate (HR), RR, PQ, QRS, and QT intervals were calculated from the ECG wave. For interbreed and interspecies comparisons, allometric body mass corrections were applied to the HR using a geometric ratio, HR/BW−0.25, and conventional human geometric ratio was considered to be 240.
8
The following QT interval corrections (QTc) were applied: QT/RR, Fredericia (QTc[F]), and Bazett (QTc[B]). In addition, a hyperbolic formula was used to estimate the correction parameters for the QT interval based on the present set of individual data in the different breeds: QTc =
Chemistry
Serum chemistries were run in one of two quality-controlled laboratories: either at Antech Diagnostics® Laboratory (Antech, Chesterfield. MO) or on a Beckman Coulter AU480 Chemistry Analyzer at SRC. Samples were obtained from 2- to 4-month-old Sinclair, 3- to 6-month-old Hanford, 3- to 6-month-old Yucatan, and 3- to 14-month-old Göttingen minipigs. Hanford and Sinclair serum chemistry testing was performed at Antech, Yucatan serum chemistries were run at either Antech or SRC, and Göttingen serum chemistries were performed at SRC.
Pathology
Gross and microscopic cardiovascular and renal pathology were performed by anatomic pathologists. Additional Göttingen histopathology was obtained from Gad et al. 7 and included for comparison purposes.
Data
Data are expressed as mean ± standard deviation, unless indicated otherwise. Parametric linear, logarithmic, and hyperbolic regression analyses were conducted using Microsoft Excel (MS Office 2013).
Results
Cardiovascular parameters
There was great variation in both HW and BW across breeds (Table 1). Overall, at 3–9 months of age, both Hanford males and females have larger HW and BW than the other breeds. On the other hand, Göttingen males had the highest heart weight to body weight ratio (HW:BW), yet females had the smallest. Other than the Göttingen females, the Hanford breed as a whole had the smallest HW:BW. When the data from the 3- to 9-month-old minipigs are compared to the findings in the older population of minipigs, it is clear that HWs and BWs increased with age, while HW:BW decreased with age. Of the breed information available at the older ages, the Hanford breed persisted in having the largest HW, but the Sinclair breed was the one with the largest BW and the smallest HW:BW. The Yucatan minipigs had the smallest BW and largest HW:BW; the age range of the Yucatan population was also younger than that of the other two breeds.
Organs weights in control minipigs.
HW: heart weight; BW: birth weight.
In comparison, KWs also demonstrated variation across the different breeds. Again, Hanford males and females had larger KWs, 148.1 ± 26.1 g and 121.9 ± 16.0 g, respectively, than the other breeds. Yucatan male and female KWs were 140.93 ± 7.55 g and 94.22 ± 9.36 g, respectively. Sinclair male and female KWs were the smallest at 58.9 ± 6.92 g and 53.2 ± 4.10 g, respectively. Likewise, kidney weight to body weight ratio (KW:BW) decreased with age as well. Sinclair males and females had KW:BW of 0.47 ± 0.10 and 0.43 ± 0.05, Yucatan males and females had KW:BW of 0.51 ± 0.08 and 0.36 ± 0.01, and Hanford males and females had KW:BW of 0.39 ± 0.05 and 0.36 ± 0.05, respectively.
In vivo vascular measurements of external iliac and femoral arteries of 15- to 19-month-old Yucatan minipigs were as follows: left external iliac: velocity of 14.82 ± 3.32 cm/s, diameter of 4.89 ± 0.34 mm, flow of 138.80 ± 32.39 ml/min; right external iliac: velocity of 16.56 ± 3.53 cm/s, diameter of 4.80 ± 0.30 mm, flow of 151.14 ± 38.82 ml/min; left femoral: velocity of 15.88 ± 3.69, diameter of 3.84 ± 0.24 mm, flow of 93.30 ± 26.92 ml/min; right femoral: velocity of 14.33 ± 2.64 cm/s, diameter of 3.93 ± 0.35 mm, flow of 87.64 ± 21.54 ml/min.
Postmortem cardiac vessel measurements were taken from Sinclair minipigs that were 2–4.7 years old, as well as 1- to 4.5-year-old Hanfords; slightly different measurements were taken from 1- to 2-year and 6- to 9-month-old Yucatans. Sinclair and Hanford minipigs had similar heart circumference and height; Sinclair had a circumference of 22.1 ± 1.85 cm and height of 13.6 ± 1.02 cm, while Hanford had a circumference of 23.1 ± 1.95 cm and height of 12.8 ± 1.30 cm. Sinclair and Hanford minipigs also had similar aorta ID and OD; Sinclair had an aorta ID of 16.43 ± 1.66 mm and an OD of 20.52 ± 1.84 mm, and Hanford had an aorta ID of 16.87 ± 3.69 mm and an OD of 21.06 ± 4.02 mm. All three breeds had LAD ID, LCX ID, and right coronary ID measured; in that order, the results were as follows: Sinclair: 1.87 ± 0.27 mm, 2.49 ± 0.82 mm, 1.47 ± 0.25 mm; Hanford: 1.70 ± 0.35 mm, 2.31 ± 0.38 mm, 1.52 ± 0.44 mm; 1- to 2-year Yucatan: 1.54 ± 0.52 mm, 1.93 ± 0.71 mm, 1.51 ± 0.42 mm; 6- to 9-month Yucatan: 1.65 ± 0.30 mm, 1.83 ± 0.45 mm, 1.91 ± 0.24 mm. The Yucatan minipigs carotid IDs are 2.48 ± 0.45 mm for the 1- to 2-year-olds and 1.62 ± 0.42 mm for the 6- to 9-month-olds.
The distribution of HR across the breeds is shown in Table 2. HR at rest ranged from 96 to 133 beats/min in the males and from 92 to 126 beats/min in the females. Some differences could be noticed between the breeds, and for comparison purposes, an allometric geometric body mass correction was applied to the HR. The mean geometric ratio was 236 with a standard deviation of 51. Interbreed comparisons indicated that the smallest ratio was found in the Göttingen male and female and the highest ratio was found in the Hanford male and female. This is in line with the observation that Göttingen minipigs had the highest HW:BW ratio.
Mean ECG results in control minipigs, 3–9 months of age.
ECG: electrocardiogram; BW: birth weight; HR: heart rate.
Electrocardiographic segment values are shown in Table 2 as well and also exhibited some variability across breeds. As expected, the largest RR intervals were found in the breeds with the lowest HR. The lengths of the different segments PR, QRS, and QT were found to be in keeping with the length of the RR interval across all breeds. The average uncorrected QT interval length was 273 ± 39 ms. The HR dependency of the QT interval was evaluated by plotting the uncorrected QT against RR (Figure 1(b)). A hyperbolic formula was the best fit (Pearson’s
(a) Lead II configuration QRS complexes electrocardiograms in pigs and dogs with comparison of the different segments (QT, QLVPend, and QS2) in relation to the electromechanical window (EMw). (b) Individual hyperbolic relationship between the RR interval and the QT interval in all breeds of minipigs.
Clinical pathology
Figure 2 demonstrates serum chemistry values commonly measured in evaluation of cardiovascular safety. The Göttingen breed exhibited slightly lower mean electrolyte values than the others but had some of the highest total cholesterol and triglyceride values as well. The Sinclair breed had a noticeably higher phosphate level than the others; otherwise, Sinclair, Hanford, and Yucatan minipigs had very similar electrolyte values.
Serum chemistry values (in mmol/L) of the four discussed breeds of minipigs; results are mean ± standard deviation.
Anatomic pathology
Among the multiple studies conducted at Sinclair and utilized as resources for anatomic pathology information, there were no recorded macroscopic pathologic findings in the hearts or kidneys. Overall, the incidence of cardiovascular background histopathologic findings was rather low as well (Table 3). One finding consistent across the breeds was renal mononuclear infiltrates of mild to moderate severity. The Göttingen breed had the lowest incidence of findings relative to the number of animals (143 males and 143 females). The Hanford breed (59 males and 60 females) had the most findings; most were various forms of inflammation, including chronic interstitial inflammation, lymphohistiocytic inflammation, and arteritis/periarteritis in the kidneys and endocarditis, myocarditis, and epicarditis of the heart. Yucatan minipigs (18 males and 21 females) had a relatively high occurrence of mononuclear infiltrates in the kidneys (33.3% of males and 52.4% of females) and a lower incidence in the heart (5.2% of males). Myriad other findings varied widely across the breeds, as listed in Table 3.
Percentage incidence of background histopathologic features in control minipigs (%).
Discussion
The use of minipigs in biomedical research has been increasing over the past decades, 9 and understanding of swine physiology and pathophysiology, including cardiophysiology, is increasing as well. 10,11 For an example, a comparison of human and swine similarities and differences focusing on the cardiovascular system and related functions is displayed in Table 4. The differences do not imply that the information on anatomical and physiological processes between swine and man may not be applied to humans. In fact, we can profit from these differences by learning more about the cardiovascular pathophysiological processes observed in humans and can extrapolate the information gleaned from minipig to man as well as to other minipigs.
A comparison of human and swine cardiovascular system and function.
EM: electromechanical.
It is widely recognized that there are hematologic, pathologic, age, and size-related background differences between the breeds of minipigs, and these background differences need to be recognizable and kept in consideration when interpreting study data. 4,12 The task of determining normal minipig cardiovascular information is well underway by many; some of these past findings were compared to corresponding results of the current study and were found to be equivalent. For example, the current Göttingen ECG findings are comparable to those in a study by Schuleri et al. 4 comparing adult Göttingen minipigs to juvenile Yorkshire pigs, as well as to those reported by Eckenfels and Schuler. 13 Additionally, Smith et al. 12 have compared cardiac function and morphology among 4-month-old Yucatan and Hanford minipigs; the Hanford breeds of their study had a smaller HW:BW than the Yucatan breeds, which is similar to the 3- to 9-month findings of the present study. Although the present study is not exhaustive by any means, these reported differences between breeds will aid investigators in selecting a relevant lineage of minipigs for specific cardiovascular parameters that may be required during drug safety evaluation.
In cardiovascular studies, since utilizing anatomy comparable to that of humans is crucial, minipig selections are often based more on size than on age or breed. According to Swindle et al., 2 the only minipig breed to develop organs completely comparable to those of humans in size are the Hanford minipigs, and they achieve this at 6–8 months of age. It is valuable, though, to have normal cardiovascular information for all breeds, detailing not only weights and organ size but also physiologic functions, background histopathology, and drug safety responses. To this extent, a comparison of cardiac function and size is shown in Table 1. As demonstrated, some variations in cardiovascular function relative to body size occur among these lineages. For example, the Hanford males had a slightly smaller HW:BW at 3–9 months of age than the other groups (Hanford: 0.41; Sinclair: 0.43; Yucatan: 0.47; Göttingen: 0.50). HW:BW along with HW and BW separately can be utilized to find a breed and an age that most closely resembles humans. The interspecies similarities between the cardiovascular systems make these lineages of minipigs suitable as models for the human counterpart. The in vitro vascular dynamics and vessel diameters, as well as the postmortem cardiac vessel measurements described earlier, are also of value, since minipigs are the ideal size for testing intra- and extravascular devices designed for humans. 5 Additionally, drug safety pharmacology as a specialized area focusing on the negative effects of drugs on key physiological systems conducts many testing procedures in vitro (ligands, isolated heart, isolated organs); however, in vivo tests are also an integral part of these assessments. Frequently, minipigs are used for these live animal studies, and the evaluation of the functional cardiovascular system, in addition to the anatomical characteristics, is a high priority.
Figure 1(a) shows a “typical” QRS complex in lead II from a pig (top) and from a dog (bottom). The pig represents a category II complex (most mammals except primates and carnivores) and the dog represents category I (primates and carnivores). 14 The differences in configurations of these QRS complexes arise principally from differences in pathways of ventricular activation; those in turn arise from differences in distribution of Purkinje fibers within the ventricular-free walls and from anastamotic branches between the left and right main bundles observed in animals of category II 15 (Figure 3). The “electromechanical window” (EMw) describes the temporal difference between these events (cf Figure 1). The EMw is calculated as the difference between the QT interval and the QVLPend interval. The EM coupling is the relationship between the duration of electrical systole (measured indirectly by the time between the Q wave and the end of the T wave; QT interval) and that of the mechanical systole (measured indirectly by the time between the Q wave and the second heart sound; QS2). In healthy animals, the duration of the QT interval is shorter than but closely parallels the duration of the QS2. Changes in autonomic tone are associated with an inversion of this normal EM coupling ratio; this ratio has been singled out as a useful indicator of several cardiovascular diseases, such as “the QT>QS2 Syndrome.” 16 Notice that the highly different configurations of the QRS complexes stem from differences in ventricular activation that stem from differences in distribution of Purkinje fibers and are underlined by differences in the EMw, leading to shorter QT and longer EMw in swine (Figure 3).
Comparative distribution of Purkinje fibers within the ventricular walls in category I animals (dog and primates) and category II animals (pig and noncarnivore mammals) with underlining differences in the electromechanical window (EMw).
Variations in cardiovascular parameters, such as the QRS complexes, do also occur among the different lineages of minipigs and should be considered when constructing experimental designs (cf Figure 1(a) and Table 2). For example, the electrophysiological heart segments duration (e.g. mean RR ranging from 488 to 655 ms and mean QT interval ranging from 262 to 302 ms) and their ratio (QT/RR) varied among the three lineages (Table 2). The same applies to the QTc, either by QTc[F] or QTc[B]. In addition, the power function analysis of the present data set of individual QT and RR intervals indicates that QTc[B] might be better fitted for the minipig than the other methods of correction (Figure 1(b)). This indicates that minipigs (and pigs) tend to be characterized by shorter QT interval when compared to the EMw but are characterized by longer relative QT when compared to human (QT: 375 ms and QT/RR: 0.452 17 ).
Even so, information obtained from the pig, with a congenitally relatively long QT interval, may be extrapolated to man. Those who study both pigs and dogs are well aware that the pig develops arrhythmias, including ventricular fibrillation, with little provocation, whereas the dog requires, at times, monumental interventions. Thus, in the evaluation of the cardiovascular effects of drugs, studies conducted on pigs might be expected to possess relatively high sensitivity compared with studies on dogs. The effects of known antiarrhythmic drugs have indeed been tested in minipigs and have demonstrated that minipigs can be used for prediction of drug-induced QT interval prolongation. 18 The similarities between their cardiovascular systems make these four lineages of minipigs suitable animals to model the human counterpart. The fundamental cardiac conduction differences lie in the nerve fiber content of the swine heart, which has a bundle richer in nerve trunks, supporting the role of a neurogenic component to conduction in swine. As indicated, EMw predicts the susceptibility to arrhythmia (EMw lengthening results in Torsade de Pointes (TdP) and EMw shortening results in absence of TdP). Therefore, swine are more prone to develop TdPs than any other species. In addition, because their diastole is relatively brief, swine are prone to coronary insufficiency. This results in increased sensitivity and decreased specificity when evaluating the effects of drugs (e.g. dofetilide, omecamtiv, mecarbil) or exercise on cardiac electrophysiology. In addition, these QRS intervals and their underlying EMw differences will aid investigators in selecting a relevant lineage of minipigs if specific cardiovascular parameters are required.
Serum chemistry results (Figure 2) are frequently used as indicators of cardiac health. Since cardiovascular health and renal health are frequently inextricably linked, renal findings are included alongside cardiovascular findings in the present study. To develop a model of hypercholesterolemia, hypercholesterolemia and hypertriglyceridemia can be induced in swine through diet. Hypercholesterolemic swine, like humans, can then develop atherosclerosis and decreased cardiac function. Heart failure results in decreased renal blood flow, which leads to renal ischemia. Subsequent damage to the glomerular epithelium leads to decreased glomerular filtration. When this decreased filtration is severe enough, phosphate is retained which causes a secondary hypocalcemia. Additionally, serum potassium levels can vary; if hyperkalemia occurs, it can in turn have serious effects on cardiac rhythm. Finally, as tubular resorption decreases with decreased renal function, sodium and chloride are lost as well eventually resulting in hyponatremia and hypochloremia. 19
Being able to use this background information to select a breed of minipigs most relevant to studies requiring similarities to humans also requires knowledge of normal human data; this can be drawn from establishment-specific references, or it can be determined based on published information. For example, according to one of the Center for Disease Control’s National Health Statistics Reports, the mean resting pulse rates in adults over 20 years of age from 1999 to 2008 were 74 beats/min for females and 71 beats/min for males. 20 Standard published human ECG results consist of an HR of 60–100 beats/min, a mean PR interval of 120–200 ms, a mean QRS interval of <120 ms, and a mean QT interval of 420 ms. 21,22 In an earlier National Health Statistics Report, the mean weight of female adults over 20 years of age in the United States from 2003 to 2006 was 74.7 kg and that of males was 88.3 kg. 23 Additionally, a study evaluating the disease-free aging heart reported BW from adults over 20 years of age ranging from 51 to 99 kg and HW of 123 to 306 g. 24
Conclusions
Enormous amounts of valuable information on cardiovascular physiology and pathophysiology have resulted from experiments conducted on minipigs. Minipigs have many anatomical and physiological differences in cardiovascular and related functions from man, but they have many similarities as well (Table 4). Because of features of pathophysiology and safety pharmacology, minipigs will remain useful for anticipating findings in man and, where differences exist, they may be exploited to understand physiology and pathophysiology in other species. Further assessments comparing the translational values of data from swine, dog, nonhuman primate, and small rodents to man may be performed from existing databases or from species to species comparisons made in retrospective studies, such as this one, or in prospective trials. Continuing to contribute background and translational data will further the value of minipigs as a continuously increasing participant of the drug safety and cardiovascular pharmacology industry.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.