Detection of early myocardial cell death in owl monkeys (Aotus nancymai) using complement component C9 immunohistochemistry in formalin-fixed paraffin-embedded heart tissues: A retrospective study

Abstract

Background:

Owl monkeys are commonly used in biomedical research which is affected by the high incidence of cardiomyopathy in this species. Occasionally, owl monkeys with no clinical signs of heart disease are found dead and at necropsy show no, or very mild, cardiomyopathy. A possible explanation for sudden death is acute myocardial infarction, however, early myocardial changes may be difficult to assess by conventional stains and light microscopy.

Methods:

Complement component C9 immunohistochemistry was performed in paraffin-embedded heart tissue samples from owl monkeys who died suddenly, or were euthanized due to sickness, to determine if these animals suffered from acute myocardial infarcts.

Results and Conclusion:

C9 deposits were found in the myocardium of 19 out of 20 (95%) animals. The findings in this study suggest owl monkeys suffer from acute myocardial infarcts and complement component C9 immunohistochemistry may be a useful diagnostic tool.

Introduction

The owl monkey (Aotus spp.) is an important animal model in biomedical research, commonly used in malaria vaccine research and other infectious diseases.^1–3 However, efforts to establish captive breeding colonies have been hampered by the high incidence of cardiomyopathy and nephropathy in these species.^4–11 Several studies suggest the hypertrophic and dilated cardiomyopathy commonly observed in captive Aotus spp. may be due to chronic essential arterial hypertension possibly engendered by an exaggerated sympathetic response to environmental events.^5,6,12 Animals with severe cardiomyopathy usually show clinical signs of heart failure and are treated symptomatically, however, the prognosis is poor since the condition, in humans as well as in owl monkeys, is usually irreversible and progressive.^9,13

Occasionally, owl monkeys with no clinical signs of heart failure are found dead during clinical rounds or die during periods of strenuous physical activity or stress and at necropsy show no, or very mild, cardiomyopathy with no organ lesions that may explain the cause of death. It has been suggested that these animals may die due to cardiac arrythmias as result of atrial and/or ventricular fibrillation. However, studies where electrocardiography was performed have not shown abnormalities that may suggest owl monkeys suffer from cardiac arrythmias.^7,8 Another possible explanation for sudden death is acute myocardial infarction. A study showed owl monkeys with proteinuria had significant higher serum creatine kinase (CK) values compared to animals without proteinuria but no significant differences in serum aspartate aminotransferase (AST) or lactate dehydrogenase (LDH) concentrations.⁶ Seven (29%) animals in the same study had CK/AST ratios suggestive of myocardial infarction but only two out of the seven animals (28.57%) showed clinical signs of heart disease.⁶ Postmortem diagnosis of acute myocardial infarction can be challenging, if death occurs minutes to a few hours after the ischemic event, mild changes like cardiomyocyte hypereosinophilia, wavy fibers, coagulative necrosis, and nuclear alterations might be subtle.¹⁴ In addition, some early pathology changes may reverse.^15,16

In humans, a definitive histologic diagnosis of acute myocardial infarct can usually be made after at least six hours of the ischemic event when karyolysis and neutrophil infiltration are observed with later evident cardiomyocyte necrosis and increasing inflammatory infiltrates.¹³ This means the individual with an ischemic insult have to live for at least six hours to have detectable irreversible changes in the myocardium by conventional light microscopy examination. A method in human pathology practice to detect acute myocardial infarcts postmortem is the nitro-blue tetrazolium test reaction. The stain will turn viable tissue dark blue while nonviable, necrotic, and fibrotic tissues, remain unstained.¹⁷ However, this method is cumbersome and can only be done with fresh tissues. In dogs this method has its limitations only allowing the detection of myocardial infarction when the ischemic event is at least 5–6 hours old.¹⁸ Cardiomyocyte death due to ischemia of shorter duration may not be detected by this method.

Special techniques have been developed in an effort to diagnose acute myocardial infarcts of shorter duration including demonstration of complement component C9 in formalin-fixed, paraffin-embedded, heart tissue samples.¹⁹ C9 is the final product of the complement cascade, part of the membrane attack complex, which attaches to the cell surface disrupting the integrity of the cell membrane causing osmotic lysis and cell death.^16,20 In humans, C9 can be detected by immunohistochemistry on affected cardiomyocytes as early as 30–60 minutes after onset of symptoms of acute myocardial infarction allowing detection of very early myocardial infarcts.²¹ In this retrospective study, we used C9 immunohistochemistry to help determine if owl monkeys suffered acute myocardial infarcts in order to better understand the etiopathogenesis of cardiomyopathy in this species.

Materials and Methods

Twenty owl monkeys (Aotus nancymai), 12 males and 8 females, were selected for this study. Out of the 20 animals, 7 (4 males and 3 females) had died suddenly with no previous history of heart disease and 13 (8 males and 5 females) were found with clinical signs suggestive of acute myocardial infarction (i.e., muscular weakness, lack of stamina, dyspnea, tachypnea, cyanosis, subcutaneous edema, convulsions, and syncope) and were euthanized. All animals were adults except for one 10-month old female. The owl monkey colony at the National Institute of Allergy and Infectious Diseases, National Institutes of Health, comprise an average daily census of approximately 200 animals. Colony history and husbandry procedures have been described in detail before.²² Briefly, the animals were housed in stainless steel 6.0 square-foot cages (Primate Products Inc., Miami, FL) with PVC nesting boxes and wood perches with a 12:12 dark/light hour photoperiod cycle and room temperature at 24° Celsius. Animals were pair housed (male/female) when possible. Standard husbandry procedures included feeding Purina New World Primate Diet 5040 (Purina Mills, St. Louis, MO), Zupreem Primate Diet Canned (Zupreem, Shawnee, KS), diet supplements (a mash prepared in-house and fruits and vegetables), and water ad-libitum. The monkeys were housed and cared for according to the “Guide for the Care and Use of Laboratory Animals”²³ and Animal Welfare Act and Animal Welfare Regulations.^24,25 All animals were enrolled in Institutional Animal Care and Use Committee-approved malaria candidate vaccine studies but not infected with malaria parasites at the time of death. Sick animals with a poor prognosis were euthanized according to the American Veterinary Medical Association Panel on Euthanasia.²⁶ Tissue samples from all major organs were collected at necropsy, fixed in neutral buffered 10% formalin, embedded in paraffin, and processed routinely for histological examination. Slides from heart samples were stained with Hematoxylin & Eosin (H&E) and Masson’s Trichrome stains per routine methods and, for complement component C9 by immunohistochemistry.

Immunohistochemistry (IHC): paraffin-embedded tissues were cut to a thickness of 5μm, placed on Probe-On Plus slides (Fisher Scientific, Pittsburgh, PA). The tissue was rehydrated, antigen retrieved with citrate buffer, pH 6.0 for 20 mins in a pressure cooker and allowed to cool to room temperature (RT). The slides were subsequently rinsed with TBST except dH2O was used for the final rinse. Slides were incubated with SNIPER (Biocare Medical, Pacheco, CA) for 15 mins and rinsed with TBS. Blocked with Peroxidazed (Biocare Medical, Pacheco, CA) for 5 minutes. Slides were incubated with rabbit anti-human complement C9 polyclonal antibody for 45 minutes (1:50, MyBioSource, San Diego, CA, catalog # MBS2401555) followed by two TBS rinses. Mach 2 (Biocare Medical, Pacheco, CA) was applied for 25 minutes and DAB+ (Biocare Medical, Pacheco, CA) was applied for 4 minutes. All slides were counter stained with CAT Hematoxylin (1:5) for two minutes. Complement C9 immunolabeling was present within many myocardial vessels serving as internal positive control. For negative controls, rabbit polyclonal IgG was used instead of primary antibody.

H&E and Masson’s Trichrome stained tissue sections were evaluated by light microscopy for myocardial damage according to the stage classification used in humans by Fishbein et al.²⁷ and Kumar et al.¹³ (Table 1) and results compared with its corresponding C9 immunohistochemical stain slide. C9 positive cells, those undergoing irreversible damage, were stained dark brown. The slides were graded for cell staining intensity and distribution within the myocardium according to the following classification: negative (no staining) = 0; weak (only visible at high magnification) = 1; moderate (readily visible at low magnification) = 2; strong (strikingly positive at low magnification) = 3.

Table 1.

Timeline of histologic changes in human myocardial infarction. Adapted from Fishbein et al.²⁷ and Kumar et al.¹³

Time	Light Microscopy
0–½ hour	None.
½–4 hours	Usually none; variable waviness of fibers at infarct border.
4–12 hours	Beginning coagulation necrosis; edema; hemorrhage.
12–24 hours	Ongoing coagulation necrosis; pyknosis of nuclei; myocyte hypereosinophilia; marginal contraction band necrosis; beginning neutrophilic infiltrate.
1–3 days	Coagulation necrosis, with loss of nuclei and striations; interstitial neutrophil infiltrate.
3–7 days	Beginning disintegration of dead myofibers, with dying neutrophils; early phagocytosis of dead cells by macrophages at infarct border; lymphocyte/plasma cell infiltrate appear.
7–10 days	Well-developed phagocytosis of dead cells; early formation of fibrovascular granulation tissue at margins; prominent lymphocyte/plasma cell infiltrate; occasional eosinophil infiltrate.
10–14 days	Well-established granulation tissue with new blood vessels and collagen deposition; “Anitschkow” type cells present at infarct border.
2–8 weeks	Increased collagen deposition, with decreased cellularity.
>2 months	Dense collagenous scar.

Results

On microscopic examination, all animals showed some myocardial changes, most of them mild or subtle and affecting small areas of the myocardium (Table 2). The most common changes noted were isolated as well as small clusters of cardiomyocyte hypereosinophilia (n=19; 95%) followed by interstitial edema and “wavy” myofibers (n=18; 90% each). Various degrees of collagen deposition and fibrosis were common (n=17; 85%) as well as nuclei pyknosis (n=15; 75%) and focal myofiber loss of nuclei and striations (n=14; 70%). Focal mild lymphocyte and plasma cell infiltrate was commonly observed (n=11; 55%) and contraction bands (n=10; 50%), followed by a few isolated cells undergoing coagulation necrosis (n=9; 45%). Less frequently, small numbers of macrophages (n=7; 35%), “Anitschkow” type cells (n=5; 25%) and mild hemorrhage (n=4; 20%) were noted. Occasional mild eosinophil infiltrates (n=3; 15%), small foci of disintegrating dead myofibers (n=3; 15%), and mild neutrophilic infiltrates (n=2; 10%) were observed. In addition, six (30%) animals presented with hyaline arteriolosclerosis of small coronary arteries with thickening of the vessel wall and narrowing of the lumen. The left ventricular subendocardium and perivascular areas appeared to be more severely affected and were characterized by multifocal loss of myocardial cells replaced by collagen and fibrous tissue. Remaining cardiomyocytes were separated by mature collagen bundles and fibrocytes and had swollen vacuolated sarcoplasm and hypertrophied nuclei. In a few cases scattered few lymphocyte and plasma cell infiltrates were observed. The most common lesion accompanying cardiomyopathy was nephropathy characterized by interstitial nephritis with arteriosclerosis (n=12; 60%). There was one case of enteritis, one case of rhabdomyolisis, one case of diverticulitis and peritonitis, and one case of bone marrow pancytopenia, enteritis, and sepsis. Examination of H&E stained myocardial sections suggests the animals had recent (hours), as well as older (weeks to months), healing and/or healed myocardial lesions as per the timeline of histologic changes described by Fishbein et al.²⁷ and Kumar et al.¹³ in Table 1.

Table 2.

Microscopic findings in myocardium of twenty owl monkeys with sudden death or euthanized.

Animal number	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
Sex: Male (M) or Female (F)	F	F	F	M	M	F	M	M	M	F	M	M	M	F	F	M	M	M	F	M
Died (D) or Euthanized (E)	E	D	D	D	E	E	D	E	D	E	D	E	E	E	D	E	E	E	E	E
“Wavy” myofibers	x	x	x	x	x	x	x	x	x	x	x	x		x	x	x	x	x	x
Coagulation necrosis			x	x		x	x		x	x		x			x					x
Interstitial edema	x	x	x	x	x	x	x	x	x	x	x	x	x	x	x		x		x	x
Hemorrhage	x	x	x									x
Nuclei pyknosis	x	x	x	x	x	x	x						x	x	x	x	x	x	x	x
Myocyte hypereosinophilia	x	x	x	x	x	x	x	x	x	x	x	x	x		x	x	x	x	x	x
Contraction band necrosis	x	x		x		x		x		x				x			x	x		x
Neutrophilic infiltrate			x																x
Loss of nuclei & striations	x						x		x	x	x	x	x	x	x	x	x	x	x	x
Disintegration of dead myofibers			x									x								x
Macrophages			x			x						x	x	x	x				x
Lymphocyte/plasma cell infiltra		x	x	x		x			x			x	x	x	x	x			x
Eosinophil infiltrate			x									x			x
Collagen deposition		x	x	x	x	x		x	x	x	x	x	x	x	x		x	x	x	x
“Anitschkow” type cells													x		x	x		x	x
Dense collagenous scar		x	x	x		x		x				x	x	x	x				x	x
Complement C9 immune stain	x	x	x	x	x	x	x	x	x	x	x	x	x		x	x	x	x	x	x

x, indicates presence/positive.

Complement C9 deposits were found in 19 (95%) of the animals and were mainly characterized by large transmural (n=7; 35%) and subendocardial (n=6; 30%) areas of C9 positive staining affecting the left ventricular free wall and interventricular wall extending sometimes to the right ventricular free wall (Figure 1A). Less frequently, intramural (n=3; 15%), diffuse multifocal (n=2; 10%), and subepicardial (n=1; 5%) C9 positive areas were observed affecting the left ventricle (Figure 1C). Occasional mild C9 positive staining was noted in auricles. In most cases staining intensity was classified as 2, characterized by large areas of C9 positive myocardial cells that could be easily observed at low magnification. All 7 (100%) animals that died suddenly had C9 deposits in myocardial cells, and 12 of the 13 animals that were euthanized (92.3%) had C9 deposits in cardiac myofibers. Out of the 8 females, 7 (87.5%) had C9 deposits in myocardial cells while, in males, all 12 (100%) had C9 positive myocardial cells. In the C9 positive cardiomyocytes the C9 deposits were present stippled and/or coarsely clumped within the sarcoplasm of individual cells (Figure 1E). In most cases the C9 positive cardiomyocytes appeared within normal limits on H&E stain but on closer examination, at high magnification, many appeared to have loss, or barely discernible, cross striations and discreet intracellular clear vacuoles (Figure 1B, 1D, 1F). In some cases, more marked changes of cardiomyocyte fragmentation were noted (Figure 1F). Cardiomyocytes with cytoplasmic hypereosinophilia, but otherwise normal architecture, were rarely associated with C9 deposits suggesting this may be a potentially reversible change. In some animals, the C9 deposits were present within the sarcoplasm of cells that were undergoing obvious degeneration and atrophy, surrounded by dense fibrous connective tissue. Myocardial cells with contraction bands were usually negative to C9 immunohistochemical staining suggesting this change occurred immediately preceding the death of the animal not allowing enough time for complement C9 deposition. There were a few C9 positive immune cells, mostly macrophages, present throughout the tissue and focally within some of the affected regions. This most likely represents phagocytized cell remnants containing C9 deposits.

Figure 1.

Figure 1(A). Owl monkey heart. Left ventricular free wall showing a transmural myocardial infarct characterized by large areas of C9 positive myocardial cells (deep brown stained cells) that are easily observed at low magnification. Complement component C9 immunohistochemistry. 40X original magnification.

Figure 1(B). Owl monkey heart. Same area as Figure 1(A) showing subtle changes characterized by slightly pale areas in myocardium and other areas showing more intense staining. H&E staining. 40X original magnification.

Figure 1(C). Owl monkey heart. Left ventricular free wall showing a large intramural myocardial infarct (deep brown stained cells) with endocardium spared. Complement component C9 immunohistochemistry. 200X.

Figure 1(D). Owl monkey heart. Same area as Figure 1(C) showing paler stained areas corresponding to C9 positive areas in Figure 1(C). Cardiomyocytes with mild cytoplasmic hypereosinophilia and normal architecture were negative to C9 deposits. Interstitial edema is also noted. H&E stain. 200X original magnification.

Figure 1(E). Owl monkey heart. Left ventricular free wall showing C9 positive cardiomyocytes with C9 deposits that are stippled and/or coarsely clumped within the sarcoplasm of individual myocardial cells. Complement component C9 immunohistochemistry. 400X original magnification.

Figure 1(F). Owl monkey heart. Same area as Figure 1(E), at high magnification, many cardiomyocytes appear to have loss, or barely discernible, cross striations and discreet intracellular clear vacuoles with some fragmented cardiomyocytes. Cardiomyocytes with mild cytoplasmic hypereosinophilia and normal architecture were negative to C9 deposits. H&E stain. 400X original magnification.

Discussion

Hypertrophic and dilated cardiomyopathy along with chronic nephropathy are the most common health conditions in captive owl monkeys.^4–11 Several studies suggest this may be due to chronic essential arterial hypertension as an exaggerated sympathetic response to environmental events.^6,12 This is further supported by studies that suggest cardiomyopathy in wild-caught owl monkeys is associated with time in captivity.⁵ The cardiac changes in the owl monkeys are usually accompanied by renal vascular changes with arteriolosclerosis of the small arteries and the afferent arteriole of the renal glomeruli,^5,6 a characteristic lesion of essential arterial hypertension in humans.^13,28 In humans, hypertension can lead not only to cardiac hypertrophy, and heart failure, but also to aortic dissection and renal failure,¹³ both conditions previously reported in captive owl monkeys.^{11, 29–31} Chronic arterial hypertension causes structural and functional changes in the wall of medium and small coronary arteries increasing the stiffness which negatively affect the physiologic function and, consequently, myocardial perfusion.³² These changes occur in response to increased systolic blood pressure in an attempt to protect the organs from baro-trauma. Small coronary and renal vasculature are particularly at risk of baro-trauma and, as hypertension progresses, small artery damage leads to impaired vasodilatation.³² The impaired vasodilatation in small coronary arteries has serious consequences, since myocardial coronary blood flow occurs mainly during diastole, contributing to myocardial ischemia which is further exacerbated in hypertrophic hearts and hearts with increased interstitial and perivascular fibrosis.³³ In addition, during hypertension, the increased systolic intramyocardial and intracavitary pressures may affect subendocardial small coronary artery tone restoration during diastole disrupting myocardial perfusion.³³ Further, the elevated systolic blood pressure increases the load to the left ventricle resulting in increased myocardial oxygen demand that cannot be met resulting in low perfusion and potential ischemia.³² The necrotic myocardial cells are replaced with connective tissue which contributes to increased myocardium stiffness and susceptibility to arrhythmias.³⁴ In addition, the activated myofibroblasts continue to secrete collagen producing adverse cardiac remodeling which eventually conduces to heart failure.³⁴

Another possible mechanism at play in owl monkey cardiomyopathy is the condition known as coronary microvascular dysfunction. This is typically the mechanism underlying myocardial ischemia in humans with angina despite completely normal coronary arteriograms but it can also trigger myocardial ischemia in several other clinical conditions, including systemic hypertension.³³ Similar to essential hypertension, the condition results from medial wall thickening with a reduced wall to lumen ratio impairing coronary vascular dilatation or producing excessive coronary microvascular constriction usually affecting the whole ventricle.³³ On histologic examination, patchy, small areas of ischemic tissue are found interspersed among otherwise normal myocardium or can also be diffuse involving most of the left ventricle, both noted in the owl monkeys in this study.³³ Transient ischemia may also result from coronary spasm and can cause myocardial infarction, arrhythmias, and sudden death.^35,36 In humans, sudden cardiac death is thought to account for almost half of all cardiac deaths and are most commonly due to ventricular arrhythmias.³⁷ The first clinical manifestation of myocardial ischemia may be sudden cardiac death due to ventricular fibrillation as the end-result of a sequence of pathophysiological abnormalities during myocardial injury.³⁸ In addition, myocardial ischemia also predisposes to atrial fibrillation during acute myocardial infarction which further exacerbates left ventricular ischemia.^38,39 Arterial hypertension and heart failure also contribute to the development and persistence of atrial fibrillation.³⁹ Sudden cardiac death has been long noted to be associated with hypertensive heart disease as a result of acute and transient myocardial ischemia.⁴⁰ Another cause of sudden cardiac death in humans is exercise-induced ventricular arrythmia, clinically manifested as exertional syncope and sudden death with a genetic basis.⁴¹ With unremarkable resting ECG, the patient develops ventricular arrythmias with exercise or by catecholamine infusion.⁴¹

Interestingly, the myocardial lesions and complement C9 deposit pattern in the owl monkeys in this study resemble the lesions produced by catecholamine toxicity in humans and animal models. Catecholamines are released from the adrenal medulla and from the central and sympathetic nervous system playing multiple functions as hormones and neurotransmitters.⁴² Increased levels of endogenous catecholamines occur during adaptation to stressful conditions, however, during chronic stress, high levels of circulating catecholamines occur which have marked inotropic stimulation enhancing cardiac contractility and heart rate increasing myocardial oxygen demand resulting in areas of functional hypoxia.⁴² This is aggravated by the oxidation products of catecholamines which produce intracellular calcium overload, direct myofiber damage, coronary spasm, depletion of high energy stores, and ventricular arrhythmias.⁴² Intravascular platelet aggregation has been suggested as an ischemia producing mechanism by catecholamines.⁴³ Sustained high levels of catecholamines can lead to major morphological cardiac alterations, similar to those produced by myocardial infarction, with cardiomyocyte cell death and progressive focal myocardial fibrosis usually accompanied by mild inflammatory infiltrates and vascular lesions.^42,43

A postmortem diagnosis of acute myocardial infarction continues to be difficult, particularly when death occurs within a few hours of the ischemic event. Myocardial cell death does not occur immediately after ischemia occurs, to observe changes by conventional staining and light microscopy the individual has to survive for at least six hours.⁴⁴ The degree and duration of the vascular occlusion, sensitivity of the cells themselves to hypoxia and available collateral blood flow, all have a marked effect on the trajectory and outcome of the myocardial infarct.⁴⁴ The earliest changes seen by electron microscopy occur within 30–60 minutes and consist of cytoplasm and mitochondrial swelling with dissolution of the cristae, loss of muscle cell contraction with stretching and elongation of sarcomeres and nuclei.⁴⁵ The contraction of the viable muscle surrounding the ischemic area further traumatizes the ischemic cells leaving patches of wavy fibers at the borders of the infarct. In addition, mild myofiber eosinophilia and, if reperfusion occurs, foci of contraction band necrosis may be found.¹⁵ All these changes have been noted in owl monkeys in the current and previous studies.^5,6

The use of Complement C9 immunohistochemistry allows the detection of very early myocardial infarcts in humans.^21,46 However, in animal models of myocardial ischemia, complement deposition in infarcted areas appears to occur later than in humans. C9 deposit in non-reperfused ischemic myocardium may take up to 6 hours in rabbits and up to 8 hours in rats, characterized by scattered deposits in single cardiomyocytes showing a later complement activation and deposition when compared to humans.^20,47–49 The utility of C9 immunohistochemical staining in humans depends on infarct age, deposits starting at 30–60 min post ischemic event, peaking at 24–48 hours, and slowly decreasing with weak or absent deposits as soon as 14 days by removal of dead cardiomyocytes by macrophages and tissue replacement by reparative fibrosis.¹⁴

In the majority of the owl monkey cases in the current study, C9 staining comprised large numbers of cardiomyocytes with a transmural or subendocardial pattern. Subendocardial cardiomyocytes are more susceptible to ischemia in humans with hypertrophic cardiomyopathy and arterial hypertension.³³ This same area is where fibrosis and collagenosis appear to occur in owl monkeys with cardiomyopathy suggesting fibrosis as a result of healed subendocardial infarcts. The presence of contraction band necrosis suggests myocardial reperfusion occurred in some animals. In a few monkeys, despite having histologic changes characteristic of myocardial infarct there were no C9 deposits in sections of the affected area. This means the cardiomyocyte changes in these animals may have been reversible or, due to interspecies variability, it may be that C9 deposition in non-reperfused ischemic myocardium may take longer in owl monkeys when compared to humans. This agrees with the findings by Mathey and collaborators who concluded that, in the absence of reperfusion, C9 accumulation occurs as a late event when most of the ischemic myocardium has probably already become necrotic.⁴⁷ Another possible explanation for the lack of C9 deposits in some sections of myocardial infarcts is that the affected area was surrounded by fibrosis delaying the deposition of complement C9.

The findings in this study suggest owl monkeys suffer from acute myocardial infarcts. The myocardial fibrosis noted in some owl monkeys with cardiomyopathy suggest these animals had “silent” myocardial infarcts that healed by reparative fibrosis, a progressive condition which aggravates myocardial ischemia, eventually leading to clinical signs of congestive heart failure and/or, possibly, acute fatal cardiac arrhythmias. The histologic changes and C9 staining pattern suggest catecholamine toxicity may be in part responsible for the cardiac lesions. In addition, the vascular changes noted further supports owl monkeys in captivity suffer from essential arterial hypertension. C9 immune staining can be used for the diagnosis of acute myocardial infarct in owl monkeys, however, it appears its deposition takes longer compared to humans. Other methods, recently reported to have earlier expression in human myocardial infarcts, like connexin 43, JunB, and cytochrome c should be explored for the early detection of myocardial infarcts in owl monkeys. The owl monkey may be a potential spontaneous nonhuman primate model for the study of essential arterial hypertension, myocardial infarction, and heart failure.