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Guidelines for Experimental Models of Myocardial Ischemia and Infarction

January 2018
AJP Heart and Circulatory Physiology 314(1132)

January 2018
314(1132)

DOI:10.1152/ajpheart.00335.2017

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CC BY 4.0

Authors:

John Canty

University at Buffalo, The State University of New York

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Myocardial infarction is a prevalent major cardiovascular event that arises from myocardial ischemia with or without reperfusion, and basic and translational research is needed to better understand its underlying mechanisms and consequences for cardiac structure and function. Ischemia underlies a broad range of clinical scenarios ranging from angina to hibernation to permanent occlusion, and while reperfusion is mandatory for salvage from ischemic injury, reperfusion also inflicts injury on its own. In this consensus statement, we present recommendations for animal models of myocardial ischemia and infarction. With increasing awareness of the need for rigor and reproducibility in designing and performing scientific research to ensure validation of results, the goal of this review is to provide best practice information regarding myocardial ischemia-reperfusion and infarction models. Listen to this article’s corresponding podcast at ajpheart.podbean.com/e/guidelines-for-experimental-models-of-myocardial-ischemia-and-infarction/.

. Comparison of different approaches with strengths and limitations for each method Approach End-Point Measurements Strengths Limitations and Pitfalls

…

. Common output measurements for in vivo MI and MI/reperfusion studies Measurement Information Provided

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. Checklist of considerations for rigor and reproducibility, modified from the ARRIVE Guidelines (168) Item Details

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REVIEW Guidelines in Cardiovascular Research

Guidelines for experimental models of myocardial ischemia and infarction

XMerry L. Lindsey,

1,2

* Roberto Bolli,

John M. Canty, Jr.,

Xiao-Jun Du,

Nikolaos G. Frangogiannis,

Stefan Frantz,

Robert G. Gourdie,

Jeffrey W. Holmes,

XSteven P. Jones,

Robert A. Kloner,

11,12

David J. Lefer,

Ronglih Liao,

14,15

Elizabeth Murphy,

Peipei Ping,

Karin Przyklenk,

Fabio A. Recchia,

19,20

Lisa Schwartz Longacre,

Crystal M. Ripplinger,

Jennifer E. Van Eyk,

and Gerd Heusch

Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center,

Jackson, Mississippi;

Research Service, G. V. (Sonny) Montgomery Veterans Affairs Medical Center, Jackson, Mississippi;

Division of Cardiovascular Medicine and Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky;

Division of Cardiovascular Medicine, Departments of Biomedical Engineering and Physiology and Biophysics, The Veterans

Affairs Western New York Health Care System and Clinical and Translational Science Institute, Jacobs School of Medicine

and Biomedical Sciences, University at Buffalo, Buffalo, New York;

Baker Heart and Diabetes Institute, Melbourne, Victoria,

Australia;

The Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology), Albert Einstein College

of Medicine, Bronx, New York;

Department of Internal Medicine I, University Hospital, Würzburg, Germany;

Center for

Heart and Regenerative Medicine Research, Virginia Tech Carilion Research Institute, Roanoke, Virginia;

Department of

Biomedical Engineering, University of Virginia Health System, Charlottesville, Virginia;

Department of Medicine, Institute

of Molecular Cardiology, Diabetes and Obesity Center, University of Louisville, Louisville, Kentucky;

HMRI

Cardiovascular Research Institute, Huntington Medical Research Institutes, Pasadena, California;

Division of

Cardiovascular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California;

Cardiovascular Center of Excellence, Louisiana State University Health Science Center, New Orleans, Louisiana;

Harvard Medical School, Boston, Massachusetts;

Division of Genetics and Division of Cardiovascular Medicine,

Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts;

Systems Biology Center, National Heart,

Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland;

National Institutes of Health BD2KBig Data

to Knowledge (BD2K) Center of Excellence and Department of Physiology, Medicine and Bioinformatics, University of

California, Los Angeles, California;

Cardiovascular Research Institute and Departments of Physiology and Emergency

Medicine, Wayne State University School of Medicine, Detroit, Michigan;

Institute of Life Sciences, Scuola Superiore

Sant’Anna, Fondazione G. Monasterio, Pisa, Italy;

Cardiovascular Research Center, Lewis Katz School of Medicine,

Temple University, Philadelphia, Pennsylvania;

Heart Failure and Arrhythmias Branch, Division of Cardiovascular

Sciences, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland;

Department of

Pharmacology, School of Medicine, University of California, Davis, California;

The Smidt Heart Institute, Department of

Medicine, Cedars Sinai Medical Center, Los Angeles, California; and

Institute for Pathophysiology, West German Heart

and Vascular Center, University of Essen Medical School, Essen, Germany

Submitted 14 June 2017; accepted in ﬁnal form 3 January 2018

Lindsey ML, Bolli R, Canty JM Jr, Du XJ, Frangogiannis NG, Frantz S,

Gourdie RG, Holmes JW, Jones SP, Kloner RA, Lefer DJ, Liao R, Murphy E,

Ping P, Przyklenk K, Recchia FA, Schwartz Longacre L, Ripplinger CM, Van

Eyk JE, Heusch G. Guidelines for experimental models of myocardial ischemia

and infarction. Am J Physiol Heart Circ Physiol 314: H812–H838, 2018. First

published January 12, 2018; doi:10.1152/ajpheart.00335.2017.—Myocardial in-

farction is a prevalent major cardiovascular event that arises from myocardial

ischemia with or without reperfusion, and basic and translational research is needed

to better understand its underlying mechanisms and consequences for cardiac

structure and function. Ischemia underlies a broad range of clinical scenarios

ranging from angina to hibernation to permanent occlusion, and while reperfusion

is mandatory for salvage from ischemic injury, reperfusion also inﬂicts injury on its

own. In this consensus statement, we present recommendations for animal models

of myocardial ischemia and infarction. With increasing awareness of the need for

rigor and reproducibility in designing and performing scientiﬁc research to ensure

validation of results, the goal of this review is to provide best practice information

regarding myocardial ischemia-reperfusion and infarction models.

Listen to this article’s corresponding podcast at ajpheart.podbean.com/e/guide-

lines-for-experimental-models-of-myocardial-ischemia-and-infarction/.

Am J Physiol Heart Circ Physiol 314: H812–H838, 2018.

First published January 12, 2018; doi:10.1152/ajpheart.00335.2017.

Licensed under Creative Commons Attribution CC-BY 4.0: ©the American Physiological Society. ISSN 0363-6135. http://www.ajpheart.orgH812

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animal models; cardiac remodeling; heart failure; myocardial infarction; reperfu-

sion; rigor and reproducibility

INTRODUCTION

Ischemia occurs when blood ﬂow to the myocardium is

reduced (129). Ischemia of prolonged duration induces myo-

cardial infarction (MI), and MI is a common cause of heart

failure (295). Ischemic cardiomyopathy is the most common

cause of heart failure and can arise from remodeling after an

acute ST segment elevation myocardial infarction (STEMI)

from multiple small nontransmural infarctions or from chronic

repetitive ischemia in the absence of infarction (15). Ischemia

can range in its extent from low ﬂow to total coronary occlu-

sion, can be of short to long duration, can be successfully

reversed by reperfusion in a timely manner or not reperfused at

all, and can induce injury or provide cardioprotection. Like-

wise, there is a diverse variety of animal models to address

each type of ischemia within this spectrum. Figure 1 shows the

range of models that reﬂect the scale of ischemia and variety of

models available to better understand how the heart responds to

ischemia and the mechanisms whereby the heart can either

adapt to ischemia or progress to failure.

Experimental models of myocardial ischemia serve two

nearly opposing aims, both worthy of investigation. The ﬁrst

aim is to provide better mechanistic insight that cannot be

obtained from a clinical situation. To achieve this aim, exper-

imental studies may be reductionist with low direct applicabil-

ity to the clinical situation (e.g., when using temporally in-

duced cell speciﬁc over- or underexpression of a gene). The

second aim is to provide mechanistic insight from an experi-

mental study for translation to the clinical situation, and for this

aim experimental models must replicate the clinical setting as

closely as possible (127).

For cardiovascular science to continue advancing, experi-

mental results should be reproducible and replicable, and

rigorous experimental design is a fundamental element of

reproducibility. Reproducibility refers to results that can be

repeated by multiple scientists and is a means of validation

across laboratories. Rigor refers to robust and unbiased exper-

imental design, methodology, analysis, interpretation, and re-

porting of results. With increasing awareness by journals and

granting agencies of the need for reproducibility and rigor in

designing and performing scientiﬁc research in preclinical

studies, the goal of this consensus article is to provide best

practice information regarding myocardial ischemia and infarc-

tion models. The strengths and limitations of the different

models are discussed, with a summary shown in Table 1. We

also address ways to incorporate Animals in Research: Report-

ing In Vivo Experiments (ARRIVE) guidelines and similar

standard operating procedures (168). The extensive reference

list provided also serves as a resource for researchers new to

the ﬁeld.

IN VITRO AND EX VIVO MODELS

Myocyte Cell Culture

Model rationale. Isolated fresh or cultured cardiomyocytes

can be used as a powerful in vitro model of ischemia-reperfu-

sion (I/R), whereby ischemia is simulated with hypoxia and

reperfusion with reoxygenation (H/R). This system allows

precise control of the cellular and extracellular environment,

notably the speciﬁc impact of hypoxia and reoxygenation on

cardiomyocytes without confounding inﬂuences of other cell

types (e.g., ﬁbroblasts, endothelial cells, inﬂammatory/immune

cells, and platelets) or circulating factors (e.g., hormones,

neurotransmitters, and cytokines).

Variables measured. The most common use of this model

system is for in vitro testing of speciﬁc factors proposed to be

involved in I/R injury or cardioprotection (31, 166, 227, 240,

244, 330). After H/R, cultured cardiomyocytes undergo apo-

ptosis, accompanied by cytochrome crelease and caspase

activation or necrosis (200, 296). Thus, assays of cell viability

are often performed to assess the role of a particular genetic or

pharmacological intervention in exacerbating or protecting the

cell from H/R-induced cell death. Viability may be measured

with a variety of assays, including lactate dehydrogenase

(LDH) release or propidium iodide exclusion as an indicator of

membrane integrity (24, 31, 58). Apoptosis is assessed with

TUNEL or annexin V staining (24, 58, 166). Mitochondrial

damage, including disruption of mitochondrial membrane po-

tential, is also a key component of cellular injury after H/R and

may be assessed using ﬂuorescent dyes, such as tetramethyl-

rhodamine methyl ester (TMRM). The loss of mitochondrial

membrane potential causes TMRM to leak from the mitochon-

dria, decreasing ﬂuorescence (24, 31). In addition, reactive

oxygen species (ROS) have been implicated in H/R injury

(275); thus, ROS production is another common assessment

(24, 58, 124, 125, 227).

More detailed analyses of cardiomyocyte responses to H/R

include assessments of morphology, contractile function (i.e.,

cell shortening), intracellular Ca

2⫹

handling, and action poten-

tials (124, 166, 207). Contractile function is an important but

often overlooked variable in H/R assays, since contraction

requires ~70% of total energy utilization within a myocyte

(182). Many H/R studies use quiescent myocytes; however,

markedly impaired recovery of myocyte function and increased

cell death result when cells are stimulated to contract through-

out the H/R protocol (207). For all variables assessed, technical

replicates on the same sample should be performed to establish

the variability of the measurement technique. Biological rep-

licates often include measurements on plates or myocytes from

the same heart/harvest. If these are to be treated as independent

samples, nvalues for both the plate/myocyte number as well as

heart/harvest number should be fully reported.

There is currently no standardized protocol for H/R in

cultured cardiomyocytes, but cardiomyocyte source and H/R

conditions must be carefully considered. Theoretically, freshly

isolated adult cardiomyocytes are the ideal gold standard for

H/R experiments (166, 207, 244), although neonatal cardiomy-

* M. L. Lindsey and G. Heusch contributed equally to this work.

Address for reprint requests and other correspondence: M. L. Lindsey, Dept.

of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 N.

State St., Rm. G351-04, Jackson, MS 39216-4505 (e-mail: mllindsey@umc.

edu).

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ocytes (58, 227, 296), cardiac progenitor cells (12), induced

pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) (24,

31), and various cell lines such as H9c2 and HL-1 have been

used (330). Of these, neonatal cardiomyocytes are most com-

monly used, due to their relative ease of isolation and robust

viability for several days in culture; however, neonatal cardi-

omyocytes are more resistant to hypoxia than adult myocytes

(236, 255), and the mechanisms of this resistance remain

incompletely resolved, which may limit interpretation of such

results when designing more translational studies (235). Adult

myocytes are preferred, because ischemic heart disease almost

exclusively occurs in adults, and fresh isolation eliminates the

potential confounding factor of phenotypic transformation in

culture. The caveat is that adult cardiomyocytes are more

difﬁcult to isolate and do not survive long in culture, impeding

longer H/R protocols or those requiring pre-H/R transfection.

Therefore, many investigators turn to neonatal cardiomyo-

cytes or cell lines (e.g., H9c2 or HL-1) if genetic modiﬁca-

tions are necessary, in which case these genetically engi-

neered cells may be an important complement to in vivo or

adult cardiomyocyte studies. In addition, adult myocytes do

not form monolayers as seen with neonatal cells, limiting

their use for studies on gap junctions and electrical conduc-

tivity. Thus, authors should carefully consider experimental

end points in the context of overall study design, because

either neonatal or adult cell sources will be the best choice

depending on the question posed. For both neonatal and

adult primary cells, isolation protocols must assure for a

cardiomyocyte-enriched population (i.e., free of ﬁbroblasts

and other cell types). For neonatal cells, the differential

attachment technique is often used, whereas gravity separa-

tion is typical for adult cardiomyocytes (161, 287). Regard-

less of the isolation and enrichment method, manual or

automated cell counting, expression of cardiomyocyte-spe-

ciﬁc markers, and visualization or quantiﬁcation of T-tubu-

lar structure should be performed to verify purity and

phenotype. Use of iPSC-CMs may overcome some of these

primary cell limitations, but the response of iPSC-CMs to

H/R has not yet been fully characterized and may depend on

their maturation state (256).

The most common in vitro conditions used to simulate in

vivo ischemia are anoxia (⬍1% O

,5%CO

,94⫹%N

) and

complete substrate depletion (serum-free, glucose-free me-

dium). There are many variations to the protocol with addi-

tional conditions that more closely mimic the ischemic heart,

such as partial hypoxia, partial or no substrate depletion,

hyperkalemia, acidosis, and use of electrical stimulation (207).

The cell type needs to be carefully considered when deter-

mining optimal control and ischemic conditions. Neonatal

cardiomyocytes and cell lines (H9c2 and HL-1) favor glu-

Ischemia

Model

Spectrum

Ex vivo

isolated

perfused

heart

In vitro

cardio-

myocytes

Angina

Stunning,

hibernation,

ischemic

cardio-

myopathy

Permanent

Occlusion

Reperfused

Ablation

Cardioprotection

Mouse Permanent Occlusion MI

7 days

Matrix cross-linking

Fibroblast apoptosis

Vascular maturation

Chemokine suppression

Fibroblast tissue deposition

Angiogenesis

Chemokine induction

Leukocyte infiltration

Proliferative phase

2-7 days

Inflammatory phase

3h-72h

Maturation phase

7-21 days

Pig 60 min ischemia + 3 h reperfusion

LV Wound Healing

Phases

4 days 4 wks

Mouse 60 min ischemia + reperfusion

40 min 3 h >6 h

Area

at risk

Rat 45 min ischemia + 2 h reperfusion

Mouse 48 h

after

cyroinjury

Wavefront progression of MI

Area

risk

Cardiac

output

Afterload

Preload

Pump

Aortic

pressure

Electrical

pacing

Pressure

volume

Inflammation

Remodeling

Scar formation

Area at risk Infarction No-reflow

Infarct size

Interventions

Preload

Fig. 1. The spectrum of ischemia encapsulates in vitro, ex vivo, and in vivo models of ischemia that range from transient to prolonged in duration with acute

to chronic consequences. The pig section (bottom left) is modiﬁed from Heusch (126). MI, myocardial infarction; LV, left ventricular;

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cose metabolism and under control conditions are often

cultured in hyperglycemic medium, which is known to

induce ROS production and cell death in adult cardiomyo-

cytes. Furthermore, neonatal cardiomyocytes are insulin

resistant and supraphysiological concentrations of insulin

are required to increase glucose uptake in these cells com-

pared with adult cardiomyocytes (201). Thus, studies focus-

ing on metabolism with H/R and/or metabolic pathology

need to carefully consider the cellular environment in both

control and ischemic conditions. In addition to the cellular

environment, the duration of hypoxia and reoxygenation is

also an important consideration, as myocyte viability not

only depends on the duration of hypoxia but also the

duration of reoxygenation (166, 244).

To summarize, the major strengths and limitations of studies

using isolated cardiomyocytes are shown in Table 1. The major

strength of H/R experiments in cultured cardiomyocytes is the

ability to control precisely the cellular and extracellular envi-

ronment, each factor present in ischemic conditions (e.g.,

hypoxia, metabolic inhibition, or acidosis) can be tested alone

and in combination to determine individual contributions to

cellular injury. Even with the most carefully designed experi-

mental protocol, in vitro conditions can never fully recapitulate

the full spectrum of I/R injury in vivo. Thus, although in vitro

experiments can be mechanistically informative and identify

new targets for intervention, it is imperative that the results are

later validated in an appropriate intact animal model. Nonethe-

less, even if there is a discrepancy between in vivo I/R and in

vitro H/R experiments, important insight is to be gained from

parallel studies. As an example, angiopoietin-like protein 4

(ANGPTL4) reduces infarct size after I/R in vivo but does not

prevent cardiomyocyte death in vitro (90). These ﬁndings

indicate that other cell types (e.g., endothelial cells, ﬁbroblasts,

or immune cells) are key to the cardioprotective effect of

ANGPTL4. Likewise, factors that prevent myocyte cell death

in culture may not reduce infarct size in vivo, suggesting that

the cardioprotective effect may be outweighed by noncar-

diomyocyte factors.

Isolated Perfused Hearts

Model rationale. The isolated perfused heart is a conve-

nient and reproducible model to test mechanisms of myo-

cardial injury and cardioprotection (14, 192). The heart is

Table 1. Comparison of different approaches with strengths and limitations for each method

Approach End-Point Measurements Strengths Limitations and Pitfalls

In vitro

cardiomyocytes

Cell viability (live-dead assay) High throughput Reductionist

Type of cell death (i.e., apoptosis) Isolate effects of hypoxia/reoxygenation on

cardiomyocytes without other cell types or

circulating factors

Cardiomyocyte viability may not predict

changes in infarct size in vivo

Adult cardiomyocyte culture technically

challenging

Isolated perfused

hearts

Infarct size per area at risk Relatively easy and reproducible Tissue edema

Left ventricular function Can study ischemia and reperfusion May not fully represent the in vivo response

Assessment of cardiac troponin I as a

secondary cardiac injury index

Accurate measure of infarct size Glucose as the sole substrate

Ample sample for biochemistry Limited stability

Compatible with NMR studies Excessive coronary ﬂow

Capacity for high throughput Reductionist

Neurohormonal factor independent

Eliminates confounding effect of intervention on

systemic blood vessels or circulating factors

Angina Regional ﬂow and function Close to clinical situation Technically complex; time and cost

intensiveMetabolism, morphology, molecular

biology, nerve activity, rhythm

Hibernation/

stunning

Regional ﬂow and function Close to clinical situation Technically complex; time and cost

intensiveMetabolism, morphology, molecular

biology, rhythm

Permanent

occlusion MI

Inﬂammation, wound healing, scar

formation, remote region myocytes,

electrical activity

In the era of percutaneous coronary intervention,

~15–25% patients are not successfully

reperfused in a timely manner (53, 104)

Does not reﬂect the reperfused MI

patient response

Robust remodeling response; large effect size

Ischemia-

reperfusion MI

Inﬂammation, wound healing, scar

formation, myocyte viability, electrical

activity

Close to clinical scenario More technically challenging surgery

Reperfusion injury can expand area of

damage

Ablation Inﬂammation, wound healing, scar

formation, myocyte electrical activity

Geometrically deﬁned lesion Nonischemic lethal injury

Infarct size/location independent of coronary

anatomy

Mechanisms of cell death different from

ischemia

Cardioprotection Infarct size per area at risk

Left ventricular geometry and function; no

reﬂow; circulating biomarkers such as

cardiac troponin I

Mouse, rat, rabbit, and pig: models of low

collateral ﬂow (measurement of regional

myocardial blood ﬂow not required)

Rat and rabbit: reliable infarct production;

relatively high survival rate

Pig: mimics humans with low collateral ﬂow

Dog: large amount of historical data; shows

effect of intervention in the setting of variable

collateral ﬂow; mimics humans with high

collateral ﬂow

Mouse: small size; substantial variability

requiring large nvalues

Rat, rabbit, pig, and dog: not high

throughput; higher cost

Rabbit, pig, and dog: potential for lethal

arrhythmias

Dog: variability in collateral perfusion

(regional myocardial blood ﬂow must

be measured)

MI, myocardial infarction.

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removed from the animal and perfused, typically with a

physiological saline solution such as Krebs-Henseleit buf-

fer. For screening drugs or interventions for protective

properties, this model is ideal, because the isolated perfused

heart is studied independently of circulating factors or

neuroendocrine inputs from other organs but retains the

function, composition, and architecture of the intact heart.

This approach is also easily amenable to biochemistry or

imaging studies in a nuclear magnetic resonance (NMR)

magnet, which can provide useful information to decipher

mechanisms of cardioprotection.

Perfused hearts can be studied in a working heart mode or in

a nonworking Langendorff mode. In the Langendorff mode,

the perfusate enters the coronary arteries to perfuse and oxy-

genate the heart, which continues to beat for several hours

(292). Heart rate and left ventricular (LV) developed pressure

are measured with a ﬂuid-ﬁlled balloon placed in the cavity of

the LV and connected to a pressure transducer as indexes of

cardiac function (physiology). The heart can be perfused at con-

stant pressure, in which case the ﬂow rate can vary, or ﬂow can be

set with a pump, in which case the perfusion pressure can vary. In

the Langendorff mode, the heart does not pump against a gradient

and does not perform external work (226). In the working heart

mode, the perfusate enters the atrium at a ﬁlling pressure set by the

operator, and the heart pumps perfusate against a hydrostatic

pressure set to different levels (30). A heart model performing

external work is technically more challenging, particularly with

smaller hearts.

In a model of global ischemia, perfusate ﬂow to the entire

heart is stopped, whereas in a regional ischemic model, a suture

is tied around a single coronary artery for occlusion. After the

ischemic period (typically 20 –40 min for rodent models),

perfusion is restarted and the heart will usually beat and

develop a lower LV developed pressure than at baseline,

reﬂecting postischemic contractile dysfunction or stunning.

Contractile dysfunction is a measure of ischemic injury but is

not synonymous with cell death. Both contractile dysfunction

and cell death often result from the same mechanisms. It is

therefore important not to infer that protection against contrac-

tile dysfunction is the same as protection against infarct size.

Variables measured. To measure cell death or infarct size, it

is necessary to reperfuse the heart for a sufﬁcient duration (at

least 60 –120 min) to wash out reductive equivalents (79, 271).

Triphenyltetrazolium chloride (TTC) is then added to the

perfusate or hearts are cut into transverse slices and incubated

in TTC solution. TTC is a dye that stains viable myocardium

red due to a formazan reaction with NADH and NADPH,

which are washed out from irreversibly injured myocardium

(81, 172), whereas necrotic tissue remains unstained and thus

appears white. Necrotic tissue area is normalized to the total

ventricular area (global ischemia) or the ischemic area at risk

for infarction (regional ischemia). For regional ischemia prep-

arations, the area at risk is measured after coronary reocclusion

and staining of the nonischemic myocardium with a dye such

as Evans blue.

The susceptibility of the heart to arrhythmias during I/R is

readily assessed through recording of an electrocardiogram

(313). Updated guidelines exist for the quantiﬁcation of such

arrhythmias (56). The isolated heart is amenable to monitoring

of intracellular ions by optical methods using ﬂuorescent indi-

cators (where the signal originates from a thin layer of epicar-

dial cells) or by NMR spectroscopy (where the signal is a

global average from the whole heart). Intracellular Na

⫹

and

2⫹

concentrations have been monitored and intracellular H

⫹

concentration (i.e., intracellular pH) has been estimated in

isolated rodent hearts perfused and subjected to I/R within the

vertical bore of the NMR magnet (288). Intracellular high-

energy phosphate (ATP and creatine phosphate) have also been

monitored by this method, and recent developments using

hyperpolarized substrates now also allow real-time analysis of

metabolic ﬂux through distinct pathways (167).

The rate of occurrence of cell death is determined by the

work that the heart performs at the time it becomes ischemic.

When global ﬂow is stopped completely, the heart will con-

tinue to beat for a short period of time and continue to consume

ATP. Reducing work at the start of ischemia is cardioprotec-

tive, and this is the basis of cardioplegic solutions. Therefore,

it is important to assure that work is similar between control

and experimental hearts, which means that heart rate and

temperature must be controlled and held constant in the dif-

ferent treatment groups (281, 292). Due to the lack of neuro-

humoral factor inﬂuences on the perfused heart, heart rate is

typically lower than in an intact animal, and it can be con-

trolled by pacing. A slight (⬍1°C) difference in temperature

can result in a large difference of infarct size. Temperature is

usually measured by a probe in the heart and controlled by

immersing the heart in a ﬂuid bath.

In the absence of blood, the reduction in the oxygen-carrying

ability of the saline perfusate results in edema and an increase

in ﬂow rate. Because the mouse has a high heart rate, it is likely

that oxygen delivery is on the edge of oxygen demand under

baseline perfusion conditions. Furthermore, Krebs-Henseleit

buffer typically contains only glucose as a substrate, whereas

the heart normally uses fatty acids as its prime substrate. Fatty

acids can be given as substrates but require the addition of a

vehicle such as BSA and also require specialized methods for

oxygenating the buffer. An advantage of the perfused heart

model is that it allows one to examine the impact of different

substrates either alone or in combinations. The effect of dif-

ferences associated with perfusion with long-chain versus

short-chain fatty acids also can be studied. Ex vivo hearts can

be readily perfused with radioactive- or stable isotope-labeled

substrates, allowing evaluation of substrate selection and me-

tabolism (167, 211, 245).

The no-reﬂow phenomenon (179) can also impact infarct

size and its measurement in isolated hearts. During ischemia

and early reﬂow, the heart goes into contracture, which restricts

ﬂow to the subendocardium. The severity of no reﬂow depends

on the severity of ischemic injury and can vary between control

and protected hearts. In isolated hearts, no-reﬂow issues can be

reduced by deﬂating the balloon in the LV for a few minutes

right at the start of reperfusion (94).

To summarize, the major strengths and limitations of the

isolated perfused heart are shown in Table 1. In consideration

for all of the above factors, it is imperative that control and

treated hearts are studied under identical conditions. Although

ischemic pre- and postconditioning were originally described

in an in vivo dog model, much of the information about the

molecular signaling pathways responsible for protection was

established in perfused heart models (128).

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IN VIVO MODELS

Chronic Coronary Artery Disease

Coronary stenosis and stress-induced myocardial ischemia:

model rationale and variables measured. Reversible episodes

of ischemia can lead to contractile dysfunction in the absence

of signiﬁcant myocyte necrosis (42). Because ischemic entities

are frequently encountered in clinical practice, the task of

understanding their pathophysiology and testing therapeutic

interventions has stimulated the development of animal models

in which acute and chronic adaptations to ischemia and the

ensuing functional recovery can be evaluated over time. Most

of these entities are consequences of either brief or chronic

episodes of ischemia, and the models developed to study them

will be discussed separately below.

Chronic stable angina is a clinical condition whereby a

patient has one or more coronary stenoses that have largely

compromised or even exhausted autoregulatory coronary re-

serve. Frequently, these limitations are partially compensated

by collateral blood ﬂow from adjacent less-compromised cor-

onary arteries such that myocardial blood ﬂow and contractile

function remain normal at rest. Stress situations such as exer-

cise, emotions, or pain, however, can precipitate acute myo-

cardial ischemia with or without typical chest pain. Chronic

stable angina in patients does not usually inﬂict global myo-

cardial ischemia but is a regional event. The acute precipitation

of myocardial ischemia requires an in vivo model where an

acute coronary stenosis can be produced to reduce coronary

blood ﬂow. Alternatively, a stable stenosis must be created

where blood ﬂow is maintained at baseline but acute ischemia

is elicited, e.g., by pacing or adrenergic activation in anesthe-

tized animals or by exercise in conscious animals (9, 93, 131).

To reﬂect the regional character of chronic stable angina,

regional myocardial blood ﬂow and regional contractile func-

tion must be measured. The standard approach to monitor

regional blood ﬂow is to use microspheres (142), which have

traditionally been labeled with radioactive isotopes (66) and,

more recently, nonradioactive colored dyes or ﬂuorescent dyes

(107, 184). Analysis of regional myocardial blood ﬂow during

acute ischemia reveals an inability of perfusion to increase

distal to a stenosis compared with normal remote myocardium

(33, 39). As coronary vasodilator reserve is exhausted, there is

a major redistribution of blood ﬂow away from the ischemic

region toward the nonischemic myocardium where metabolic

vasodilation prevails. In addition, there is a transmural blood

ﬂow redistribution from the ischemic subendocardium toward

the subepicardium (93).

The gold standard for experimental regional contractile

function measurements is sonomicrometry (265). Simultane-

ous measurements of regional myocardial blood ﬂow and

regional contractile function provide a means to determine the

quantitative relationship between regional blood ﬂow (as a

surrogate for oxygen/energy supply) and regional contractile

function (as a surrogate for oxygen/energy demand). The

relation between regional contractile function and subendocar-

dial perfusion (ﬂow-function relation) demonstrates close cou-

pling during steady-state ischemia at rest as well as over a wide

range of cardiac workloads (33, 35, 37, 91, 309). During

steady-state acute myocardial ischemia, there appears to be no

imbalance between supply (blood ﬂow) and demand (func-

tion); rather, there is a matched reduction in both parameters

(129, 130). Such matching between regional blood ﬂow and

contractile function also persists during major changes in heart

rate, when both blood ﬂow and contractile function are nor-

malized for a single cardiac cycle (92). This matching can

persist for several hours and contribute to the maintenance of

myocardial viability and full recovery of contractile function

after eventual reperfusion (212). More speciﬁcally, the hall-

marks of short-term myocardial hibernation are a perfusion-

contraction match (259), together with metabolic signs of

adaptation to ischemia (210, 267) and the potential to recruit an

inotropic reserve in the dysfunctional myocardium (267). Also,

all pharmacological interventions to attenuate acute myocardial

ischemia (e.g., by nitrates, ␤-blockers, Ca

2⫹

antagonists, or

their combinations) operate along a ﬁxed ﬂow-function rela-

tionship (213–215). The two major mechanisms that precipi-

tate myocardial ischemia and must therefore be pharmacolog-

ically addressed are tachycardia (112, 113) and coronary va-

soconstriction (134, 273).

Studies evaluating brief total coronary occlusions can be

conducted in a variety of species. In contrast, to study coronary

artery stenosis, the animal under study must be large enough

that coronary artery instrumentation along with regional ﬂow

and function measurements is feasible (i.e., in dogs and pigs

that can be instrumented with a hydraulic occluder on the

coronary artery). Acutely anesthetized animals have provided

insight into short-term coronary ﬂow regulation over minutes

to hours but cannot evaluate the effects of chronic coronary

stenosis on long-term microvascular remodeling and collateral

growth over days to weeks (285). Acute experiments have the

advantage that sequential myocardial biopsies can be taken for

the analysis of metabolic and molecular analyses (101, 174,

210, 283). Microdialysis probes can be implanted to evaluate

interstitial mediators (209, 270), and the activity of the cardiac

innervation can be measured (132).

A signiﬁcant experimental challenge is maintaining a ﬁxed

degree of coronary artery narrowing throughout a study using

a hydraulic occluder. This limitation can by circumvented by

perfusing the coronary artery at constant pressure from a

reservoir, controlling ﬂow with an extracorporeal pump or

perfusion of the region of interest with an extracorporeal

pressure control system (38). A major limitation of studies in

acutely anesthetized animals are the substantial confounding

effects of anesthesia and neurohormonal activation on hemo-

dynamics and ﬂow, which alter coronary autoregulation and

produce varying degrees of coronary vasodilation and vaso-

constriction that modulate autoregulatory responses (33, 36,

39). The limitations of acute studies can be circumvented by

studying conscious, chronically instrumented animals.

While strict control of hydraulic occluders and coronary

collaterals stimulated by repetitive ischemia and chronic

stenosis at ﬁrst seems a limitation, these factors can be

capitalized upon by provoking coronary collateral develop-

ment to the point where the artery can be totally occluded

without reducing resting myocardial perfusion. This is typ-

ically accomplished in dogs using ameroid occluders, which

are hygroscopic and gradually swell to produce a progres-

sive stenosis resulting in a total occlusion within 3– 4 wk.

Collateral blood vessel growth can also be stimulated in

dogs by repetitive brief coronary occlusions using a hydraulic

occluder (303, 320). Once collaterals are developed, variability

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in the hydraulic stenosis severity is no longer a problem, and

intervention effects on stress-induced ischemia can be studied

under multiple conditions. While pigs can also develop collat-

eral-dependent myocardium after ameroid occluder placement

(260), collaterals grow more slowly than in dogs, and pigs

frequently develop a subendocardial infarction (230). The

admixture of infarcted and normal myocardium greatly com-

plicates measurements of perfusion and function. Infarction

can largely be circumvented by employing a ﬁxed diameter

stenosis on the coronary artery of farm-bred swine, resulting in

a much more severe limitation of subendocardial ﬂow reserve

than in dogs, and there is usually contractile dysfunction at rest

(74, 77). While this is an extremely useful model to study

chronic vascular adaptations and interventions to promote

angiogenesis, alterations in myocardial physiology can com-

plicate the interpretation of ﬂow changes. A major drawback of

chronic large animal models of coronary stenosis and collat-

eral-dependent myocardium is their expense and the labor-

intensive nature of the animal care and handling. The guinea

pig has a very well-developed collateral circulation that pre-

vents infarction from occurring following occlusion of a single

main coronary artery; to obtain infarction, multiple coronary

arteries need to be ligated (216). Thus, guinea pigs are not

suitable for in vivo ligation studies but can be used for heart

perfusion with global ischemia experiments. These issues have

motivated studies to assess ﬂow regulation using repetitive

coronary occlusions in rats and mice, including genetically

altered animals (303).

Coronary microembolization: model rationale and variables

measured. Subclinical atherosclerotic plaque rupture or erosion

that does not result in complete thrombotic occlusion of the

coronary artery but leaves a residual blood ﬂow into the distal

coronary microcirculation occurs spontaneously, with or with-

out clinical symptoms. Coronary microembolization is also

induced iatrogenically during percutaneous coronary interven-

tions. Atherosclerotic debris from the culprit lesion, together

with thrombotic material, soluble vasoconstrictors, as well as

thrombogenic and inﬂammatory substances, is washed into the

coronary microcirculation where it causes microvascular ob-

struction with resulting patchy microinfarcts and an inﬂamma-

tory reaction (135, 173). The inﬂammatory response includes

increased expression of tumor necrosis factor-␣associated

with profound contractile dysfunction and upregulation of

signal transduction pathways involving nitric oxide, sphin-

gosine, and ROS, which contribute to impaired excitation-

contraction coupling (32, 299). Repetitive coronary microem-

bolization can result in global LV dysfunction and, even in the

absence of overt infarction, in heart failure (261). Coronary

microembolization can be simulated experimentally by intra-

coronary infusion of inert particles of various diameter (67)

and also by intracoronary infusion of autologous microthrombi

(191). When the target under study is ischemic heart failure,

repeated coronary microembolization can be used in both small

and large animal models. When the target under study is a

spontaneous or periprocedural minor infarction, the animal

must be large enough such that regional myocardial measure-

ments of ﬂow, contractile function, metabolism, and morphol-

ogy are possible (i.e., dog or pig models are preferable).

The major strengths and limitations of angina models are

shown in Table 1.

Stunning, Hibernation, and Ischemic Cardiomyopathy

Stunning: model rationale and variables measured. When

ischemia caused by a total coronary occlusion is brief (e.g., as

may be experienced from coronary vasospasm), regional con-

tractile dysfunction persists for hours after reperfusion but then

completely normalizes within 24 h. This phenomenon was ﬁrst

demonstrated after a 15-min circumﬂex coronary artery occlu-

sion in chronically instrumented dogs, was subsequently called

stunned myocardium, and is common in patients with acute

coronary syndrome (17, 19, 143). Most investigators assume

that the complete normalization of function, lack of evidence

of infarction by TTC staining, and lack of sarcolemmal dis-

ruption on electron microscopy indicate that no cardiomyocyte

death is associated with stunning. While pathological evidence

of myocyte necrosis is indeed absent, TUNEL staining per-

formed 1 h after reperfusion demonstrates that programmed

cell death or myocyte apoptosis develops in rare isolated

cardiac myocytes and circulating cardiac troponin I is in-

creased (314). Thus, while there is no evidence of infarction in

stunned myocardium, regional myocyte loss can develop when

stunning becomes repetitive.

Because the essence of stunned myocardium consists of

relatively rapid (24 –48 h) reversibility of contractile dysfunc-

tion in the absence of TTC or pathological evidence of infarc-

tion, most studies use chronic large animal models in which

serial measurements of function can be performed. In addition,

regional ischemia is the preferred model to allow assessment of

the remote nonischemic regions of the heart as an internal

control. While stunned myocardium occurs after demand-

induced ischemia distal to a coronary stenosis (144), most

studies have used transient total coronary occlusion. Animals

are instrumented with a hydraulic occluder to produce brief

ischemia 1–2 wk after recovery from surgical instrumentation.

To assess regional function, most studies have used sono-

micrometry for direct measurements of subendocardial seg-

ment length shortening or wall thickening. Recent studies have

used transient occlusion of the left anterior descending coro-

nary artery (LAD) using a balloon angioplasty catheter in

closed-chest sedated animals where regional function can be

assessed with imaging approaches such as echocardiography

(314). The latter approach circumvents the need for chronic

surgical instrumentation through a prior thoracotomy. Echo-

cardiography can also be employed to assess stunning in mice

chronically instrumented with an occluder to produce transient

ischemia (63).

The dog (20, 143), pig (291, 314), and rabbit (18) are the

most commonly used species to study myocardial hibernation.

Pigs and rabbits offer the advantage of having little or no

collateral circulation, so that the severity of the ischemic insult

and of the subsequent contractile dysfunction are more uni-

form. In contrast, dogs exhibit a highly variable degree of

collateral circulation resulting in widely different degrees of

myocardial stunning (20). There are also species differences in

the time course of recovery despite similar occlusion durations

(277). Many studies have also used open-chest animal models,

although the severity of myocardial stunning in these prepara-

tions is signiﬁcantly exacerbated versus conscious animal mod-

els (21, 304). Most experimental approaches to assess stunning

are quite straightforward, although ventricular ﬁbrillation can

develop. This is more common in swine as opposed to canine

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models, because pigs have little innate coronary collateral ﬂow

(164). Because myocardial function assessed using segment

shortening and wall thickening is load dependent, it is impor-

tant to ensure that heart rate, systolic blood pressure, and LV

end-diastolic pressure remain reasonably constant over the

time frame of the measurements. An advantage of chronic

models using regional ischemia is that each animal can poten-

tially serve as its own control; hence, it is possible to use the

same animals to study the effects of pharmacological interven-

tions on physiological end points.

Short-term hibernation: model rationale and variables

measured. A prolonged episode of moderately severe ischemia

can be sustained for a period of hours in the absence of

pathological evidence of infarction. This phenomenon is

termed short-term hibernation (139, 212). An approximate

50% reduction in perfusion leads to reduced function and

perfusion-contraction matching, which largely prevents irre-

versible myocyte injury. If the heart is reperfused within a few

hours, contractile dysfunction persists in a fashion similar to

stunned myocardium but with a more protracted time course of

recovery (i.e., lasting in the timeframe of days rather than

hours) as is typically seen with stunning after a brief total

occlusion. This in part appears to relate to reversible myoﬁ-

brillar disassembly and myolysis in the absence of sarcolem-

mal disruption (279). Unfortunately, when the initial adaptive

response to moderate ischemia in short-term hibernation is

present for longer than 12 h, progressive myocardial necrosis

begins to develop, resulting in some degree of myocardial

infarction usually conﬁned to the subendocardium (48, 185,

269). While imposition of acute moderate ischemia was ini-

tially proposed as a mechanism of chronic hibernating myo-

cardium, the development of progressive infarction when ﬂow

reductions last longer than 12 h leads to a pathological entity

with myoﬁbrillar disassembly and cardiac biomarker release

that can no longer be deﬁned as hibernation but, rather, is more

in line with subendocardial infarction (48, 279).

Studies of short-term hibernation usually require closed-

chest animal models, although considerable insight about ad-

justments between ﬂow and function has been gleaned from

open-chest studies of myocardial metabolism (137, 210, 239).

The latter studies usually use a cannulated branch of the left

coronary artery perfused at constant pressure. Closed-chest

animal studies usually employ chronically instrumented dogs

or pigs. In these studies, a hydraulic occluder is placed around

a coronary to reduce ﬂow or coronary pressure to a ﬁxed level,

which is released after several hours.

Chronic hibernation and stunning: model rationale and

variables measured. While both stunning and short-term hi-

bernation are characterized by complete functional recovery,

chronic contractile dysfunction can develop when recurrent

ischemia develops before functional normalization (21).

Chronic contractile dysfunction from repetitive ischemia de-

velops in the absence of histological infarction, and both

hibernation and stunning involve the loss of myocytes via

apoptosis in a fashion similar to what happens after brief

episodes of ischemia (193, 314). Unlike stunning, which was

initially an experimental observation that later became associ-

ated with multiple clinical correlates, chronic hibernating myo-

cardium was ﬁrst characterized in patients with chronic isch-

emic heart disease displaying regional contractile dysfunction

in the absence of manifest ischemia (23, 139). Only later were

animal models used to identify cellular and molecular mecha-

nisms responsible for the adaptive responses to chronic isch-

emia (77).

While it was originally controversial whether or not ﬂow

was reduced or normal at rest (34), it is now clear that chronic

repetitive ischemia initially results in contractile dysfunction

with normal resting ﬂow or chronic stunning (73, 278). When

this situation persists, the reduction in function leads to a

secondary reduction in regional energy utilization accompa-

nied by reduction in resting ﬂow (76). Thus, the reduction in

resting ﬂow characteristic of chronic hibernating myocardium

is a result, rather than cause, of regional dysfunction.

In contrast to models of short-term ischemia, animal models

of hibernating myocardium are based on chronic coronary

stenoses that frequently progress to total occlusion and collat-

eral-dependent myocardium. In studies using ameroid occlud-

ers that gradually swell to produce chronic stenosis, dogs

usually do not develop contractile dysfunction at rest but can

do so when preexisting epicardial collaterals are ligated at the

time of instrumentation (36). Swine ameroid occluder models

frequently have contractile dysfunction in collateral-dependent

myocardium, and this is usually associated with some degree

of subendocardial infarction (230).

A more consistent model of hibernating myocardium can be

produced by instrumenting juvenile swine with a ﬁxed diam-

eter stenosis (1.5-mm diameter) on the proximal LAD (73, 77).

As the animals grow over the subsequent 3 mo, there is a

slowly progressive limitation in coronary ﬂow reserve, because

the LAD stenosis limits maximal myocardial perfusion, while

the mass of myocardium distal to the stenosis increases in

parallel with cardiac growth. As a result, there is a more

prolonged and gradual stimulus for coronary collateral devel-

opment so that LAD occlusion almost always develops in the

absence of infarction.

After 3 mo, regional contractile dysfunction with mild re-

ductions of resting ﬂow in the absence of infarction is consis-

tently manifest and is similar to the changes seen in humans

with hibernating myocardium caused by a chronic LAD occlu-

sion (308). Serial studies of this animal model have demon-

strated that the heart progressively adapts from a state of

contractile dysfunction with normal resting ﬂow (chronic stun-

ning) to a state where resting ﬂow decreases, consistent with

hibernating myocardium (41). Such chronic hibernation is

associated with a downregulation in mitochondrial metabolism

and regional myocyte hypertrophy that maintains myocardial

wall thickness constant in the setting of regional apoptosis-

induced myocyte loss.

Over longer periods of time (up to 6 mo) the adaptive

response of hibernating myocardium persists unchanged (75),

and the downregulation in metabolism and upregulation of

proteins involved in cellular survival and cytoprotection pre-

vent cell death and, hence, further myocyte loss (62). While

infarction does not develop in this model, revascularization

only partially reverses myocardial dysfunction and does so

over a much longer time frame than seen with either myocar-

dial stunning or short-term hibernation (237). Chronic contrac-

tile dysfunction in the absence of infarction can also be induced

using a hydraulic occluder to produce an acute stenosis in

chronically instrumented animals. Chronic stunning can de-

velop in swine subjected to daily episodes of short-term hiber-

nation (169). A more rapid transition from chronic stunning to

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hibernating myocardium than the one observed in the ﬁxed

diameter stenosis model can be achieved by acutely imposing

a critical stenosis on the LAD (301). The latter model can

develop reductions in ﬂow with regional contractile dysfunc-

tion after 2 wk of a stenosis sufﬁcient to prevent reactive

hyperemia.

The ﬁxed diameter chronic stenosis model is advantageous

in that hibernating myocardium develops reproducibly in a

predictable time frame. A limitation of the ﬁxed stenosis

porcine model is that it requires cardiac growth to produce

a progressive physiological impairment in maximum myocar-

dial perfusion, and the 3- or 4-mo period required to develop

hibernating myocardium is viewed as cost prohibitive. This

model has so far only been studied in juvenile farm bred swine

and may produce variable results if cardiac growth is attenu-

ated by limiting feeding. It is not clear whether the model can

be effected in purpose-bred swine and, particularly, in mini-

swine, where growth rates are substantially attenuated. An

additional disadvantage is that the chronic stenosis model is

associated with a high rate of spontaneous ventricular ﬁbrilla-

tion (40). This has provided insight into the mechanisms of

sudden cardiac arrest in chronic coronary disease but reduced

the success of studying chronic adaptations to ischemia in

survivors. Finally, because of the long duration of the studies

in the presence of animal growth, it is not feasible to chroni-

cally instrument animals. Nevertheless, it is feasible to use

telemetry to assess chronically LV pressure and arrhythmias in

untethered conscious animals (242).

Ischemic cardiomyopathy: model rationale and variables

measured. Ischemic cardiomyopathy is the underlying cause of

LV dysfunction in two out of every three patients with heart

failure (105). Ischemic cardiomyopathy in humans can arise

from LV remodeling after a large myocardial infarction but,

more commonly, is the result of extensive multivessel coronary

artery disease with modest amounts of diffuse ﬁbrosis and

patchy infarction in multiple coronary artery distributions (15).

Along these lines, preclinical studies have established that

chronic coronary artery stenosis can induce signiﬁcant myo-

cyte loss with modest global replacement ﬁbrosis that leads to

global LV dysfunction and varying degrees of congestive heart

failure when the area at risk is large. Conceptually, the stenosis

does not limit blood ﬂow at rest. Rather, by reducing maximal

perfusion in response to stress, it sets the stage for repetitive

episodes of subendocardial ischemia. A key feature of all

animal models of ischemic cardiomyopathy is that the myo-

cardium at risk of repetitive ischemia represents a large portion

of the LV (⬎70% of LV mass). This has been achieved using

stenosis of the left main coronary artery in rodents or multi-

vessel coronary artery stenoses in large animals. As a result of

the large area at risk, myocyte cell death arises from both

ischemia and myocyte stretch and slippage from increased LV

end-diastolic pressure (possibly also reﬂecting transient isch-

emia).

In rats, ischemic cardiomyopathy can be induced by produc-

ing a ﬁxed coronary stenosis of ~50⫺60% diameter reduction

on the proximal left coronary artery, which causes variable

degrees of LV dysfunction (44, 45). While replacement ﬁbrosis

occurs in these animals, it is patchy and modest, only increas-

ing twofold over control for an average of ⬍10% of LV

cross-sectional area. Interestingly, the degree of LV dysfunc-

tion is primarily related to myocyte cell loss (necrosis and

apoptosis) and the elevation in LV end-diastolic pressure

related to ﬁbrosis. A similar model of ischemic cardiomyopa-

thy has also been obtained in mice (189). While rodent models

afford the ability to perform higher throughput studies and use

transgenic animals to study molecular mechanisms, they have

relatively high surgical and postoperative mortality. In addi-

tion, there is considerable variability in physiological out-

comes, such that frequently animals are retrospectively cate-

gorized into mild, moderate, and severe heart failure groups.

Reproducibility of ischemic cardiomyopathy models, there-

fore, is indeed a concern.

While left main coronary stenosis is not feasible in large

animals, multivessel coronary stenoses can produce a large

ischemic risk area and recapitulate ischemic cardiomyopathy.

When ﬁxed diameter occluders are placed on both the proximal

LAD and circumﬂex arteries in growing farm-bred swine, LV

ejection fraction declines with elevated resting LV end-dia-

stolic pressure (74), consistent with compensated LV dysfunc-

tion and no overt evidence of heart failure. These animals also

exhibit primary myocyte loss with only an approximately

twofold increase in extracellular matrix accumulation. A sim-

ilar condition has been produced using multivessel ameroid

occluders in dogs (80). Aside from requiring survival surgery,

the major disadvantage of these approaches arises from the

development of sudden cardiac arrest, which in swine is related

to both ventricular ﬁbrillation and to a lesser extent bradyar-

rhythmias. In mice, a state of ischemic cardiomyopathy can be

induced using repetitive brief coronary occlusions, and this

model is associated with substantial but reversible ﬁbrosis of

the myocardial region subjected to repetitive ischemia (63).

Noninvasive imaging. Noninvasive cardiac imaging technol-

ogies such as echocardiography, magnetic resonance imaging

(MRI), and computed tomography can measure regional and

global contractile function and are increasingly available for

preclinical studies, particularly in larger animals. NMR spec-

troscopy can provide information on cardiac energetics (114).

More sophisticated imaging technologies such as positron

emission tomography can measure regional myocardial perfu-

sion and regional myocardial metabolism and sympathetic

innervation and are increasingly used in preclinical studies (77,

187, 268). MRI can serially measure myocardial perfusion

(264) and can provide reliable measurements of infarct size and

microvascular obstruction. MRI-derived edema, however, is

time dependent and sensitive to cardioprotective interventions

(141, 148). Therefore, MRI-derived edema can be used to

stratify an ischemic/reperfused myocardial region for protocol

assignment but not for quantitative normalization of infarct

size to area at risk.

To summarize, the major strengths and limitations of stun-

ning, hibernation, and ischemic cardiomyopathy models are

shown in Table 1.

Myocardial Infarction Models: Permanent Coronary Artery

Occlusion with Nonreperfused and Reperfused Myocardial

Infarction

MI: general considerations. Coronary occlusion causes im-

mediate cessation of aerobic metabolism in the ischemic myo-

cardium, leading to rapid ATP depletion and metabolite accu-

mulation and resulting in severe systolic dysfunction within

seconds (86). If the duration of the ischemic insult is ⬍15 min

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in larger mammals such as dog and pig, restoration of ﬂow

reverses the early ischemic cardiomyocyte changes (transient

mitochondrial swelling or glycogen depletion) and all cardio-

myocytes in the ischemic area can survive (158). Longer

periods of ischemia cause death of an increasing number of

cardiomyocytes. A 20- to 30-min interval of severe ischemia is

sufﬁcient to induce irreversible changes in some cardiomyo-

cytes of the subendocardial area, inducing sarcolemmal disrup-

tion and striking perturbations in mitochondrial architecture,

such as ultrastructural evidence of amorphous matrix densities

and severe mitochondrial swelling (156). These early ultra-

structural alterations mark cardiomyocytes that cannot be sal-

vaged and will ultimately die in the infarct environment (157).

Experimental studies in the canine model of MI demonstrate

a transmural heterogeneity in the myocardial response to isch-

emia, suggesting that the subendocardium, where myocardial

oxygen demand is greatest, is more susceptible to ischemic

injury than the midmyocardium and subepicardium (2). Thus,

the prevailing paradigm suggests a wavefront of cardiomyo-

cyte death that progresses from the more susceptible subendo-

cardium to the less vulnerable subepicardium as the duration of

the ischemic insult increases (159, 252). Experimental studies

in large animal models have demonstrated that ischemic myo-

cardium cannot be salvaged by reperfusion after6hofcoro-

nary occlusion (251). The increased vulnerability of subendo-

cardial regions to coronary occlusion may reﬂect a greater

reduction of the subendocardial blood ﬂow due to transmural

differences in vascularization (2, 25) and extravascular com-

pression (68, 286). The wavefront concept of ischemia devel-

oping into infarction was derived from experimental studies in

dogs, where a substantial coronary collateral circulation inﬂu-

ences the time course of cardiomyocyte necrosis (86).

The major species difference in the MI response lies in the

temporal and spatial kinetics of events and differences due to

myocardial size. In mice, durations of coronary occlusion

exceeding 60 –90 min are considered irreversible, and inﬂam-

mation and wound healing processes are accelerated (64, 88,

221, 222). In mouse and rat models, reperfused infarcts are

typically midmyocardial, and subepicardial and subendocardial

regions are relatively spared (50, 69, 325). Studies in a sheep

model of reperfused infarction also suggest that the midmyo-

cardium may be most vulnerable to ischemic injury; in con-

trast, the subendocardium is relatively resistant (263). The pig

model of coronary occlusion-reperfusion comes closest to

human STEMI in its temporal and spatial development, but

other models are nevertheless useful to study fundamental

mechanisms of MI (140).

MI: technical considerations. Extensive protocols providing

technical details for performing permanent occlusion MI and

reperfused MI in mice and rats are available (221, 222, 228,

317, 327). While MI is most commonly performed in rodent

models, protocols in other animal models are also available

(151, 183, 218, 331). For mice, the quality of open-chest

surgery to induce coronary occlusion directly impacts study

outcomes (152, 221, 222). Minimizing the size of the thora-

cotomy and limiting bleeding by entering the thorax through

intercostal muscles are recommended.

Biomarkers that have been used to evaluate the presence of

MI include cardiac troponins and creatine kinase, and plasma

proteins such as macrophage migration inhibitory factor can

also be measured as indices of injury (47, 55). Infarct size is

widely measured as a key variable for testing genetic or

therapeutic intervention efﬁcacy, and infarct size measure-

ments taken serially at both early and late time points can

evaluate the extent of infarct expansion (22). Echocardiogra-

phy can also be used for infarct sizing, with the caveat that

echocardiography does not distinguish between reversible isch-

emic dysfunction (stunning) and irreversible loss of function

and therefore a secondary method is needed for conﬁrmation of

infarct size at early time points. For more details on measuring

cardiac function in mice, the reader is advised to consult the

article Guidelines for measuring cardiac physiology in mice

(196).

Permanent occlusion MI: model rationale and variables

measured. Permanent coronary occlusion is a relevant animal

model of acute STEMI reﬂective of patients who, due to

contraindications or logistic issues, do not receive timely or

successful reperfusion (53, 104). Permanent coronary occlu-

sion yields acute ST segment elevation infarction with robust

myocardial inﬂammation and long-term remodeling, thus pro-

viding a large effect size that reduces the sample size needed to

detect differences between groups. Infarction assessed in the

ﬁrst 1–14 days after coronary ligation is histologically charac-

terized by coagulation band necrosis with a fulminant inﬂam-

matory inﬁltrate in the infarct and border zone regions. Infarc-

tion is geometrically and physiologically characterized by wall

thinning, increases in LV dimensions and volumes, and de-

creases in fractional shortening and ejection fraction.

Changes that occur over the ﬁrst week provide information

on myocyte cell death and infarct development, inﬂammation

and leukocyte physiology, extracellular matrix (ECM) turnover

and ﬁbroblast activation, and the role of endothelial cells in

neovascularization (83, 154, 165, 194, 205). Chronic evalua-

tion at time points 4 –8 wk post-MI provides information on

long-term remodeling. Whether the infarct region or remote

region is the focus of investigation depends on the question

asked. Examining the infarct region provides details on active

inﬂammation and scar formation, while examining the remote

region provides details on still-viable myocytes within the

myocardium and remote inﬂammatory and ECM processes.

Perioperative and postoperative survival should be assessed,

and the time point of delineation between these two phases

should be deﬁned. For some laboratories, the perioperative

phase includes the time until the animal recovers and becomes

ambulatory (usually within 1–3 h for mice). For other labora-

tories, the perioperative phase includes the ﬁrst 24 h after

surgery. Perioperative death within 24 h post-MI in mice is

usually due to surgical errors (or very large infarct sizes), and,

in established laboratories, the 24 h surgical mortality rate due

to technical issues is ⬍10%. In the permanent occlusion MI

model in mice, postoperative death (deaths at ⬎24 h time

point) typically occurs during days 3–7 post-MI and is due to

rupture, acute heart failure, or arrhythmias (59, 98, 233).

Autopsy is strongly recommended for all mice that die prema-

turely, to evaluate early deaths due to technical issues and later

deaths due to complications of MI. Seven-day postoperative

mortality rates are ~10 –25% (75–90% survival) for female

young mice and 50 –70% (30–50% survival) for male young

mice (47, 61, 89, 98, 152, 170, 195, 202, 206, 234, 310 –312,

319, 323). Immediate survival from the surgery can also be

affected by baseline characteristics such as obesity, diabetes, or

high levels of circulating inﬂammatory cells, which, in turn,

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determine the response to anesthesia and surgery (60, 123, 202,

203). While there is no difference in infarct tolerance between

young and middle-aged mice (323), older mice may survive

better than younger mice (202, 319).

For permanent occlusion MI models, infarct size must be

measured in fresh LV slices at the time of necropsy by TTC

staining and typically ranges from 30% to 60% of the total LV

(47, 48, 61, 89, 98, 152, 195, 202, 206, 223, 310 –312, 319, 323).

The method for calculating infarct size varies across laboratories.

Some laboratories use area calculations, other laboratories mea-

sure length in the midmyocardium, and other laboratories measure

and average lengths in the subendocardium and subepicardium.

There is no need to use Evans blue for area at risk assessment in

permanent coronary occlusion models that pass the point from

ischemia to infarction, as the entire area at risk is infarcted. It

is important that MI surgical success is conﬁrmed and that the

initial infarct injury is comparable across groups, to assess

remodeling differences at later stages. In mice, ligating the

coronary artery at the same anatomical location across groups

is important; 1 mm distal to the left atrium is the recommended

site to generate large infarcts (35– 60% of total LV). Failure to

induce MI can occur, usually due to missing the coronary

artery during the ligation step. Monitoring the electrocardio-

gram for ST segment elevation during the procedure reduces

this possibility. Echocardiography at 3 h after coronary occlu-

sion can be used to exclude animals with excessively small or

large MI before randomizing groups (153, 155, 195). Late

gadolinium-enhanced MRI is also useful for selecting animals

with consistent infarct sizes (262). When assessing effects of

treatments initiated post-MI, it is important to show that infarct

size is not different between groups before treatment. Plasma

sampling at 24 h post-MI can be used to assay cardiac bio-

markers, such as troponins and inﬂammatory cytokines, with

the caveat that these measurements can indicate presence or

absence of infarct and not extent of injury. After coronary

occlusion, care should be taken in performing these assess-

ments to minimize disturbing animals at times when cardiac

rupture may be triggered by stress, particularly at days 3–7

post-MI in untreated controls (96, 98). Small infarcts may

reﬂect technical issues in missing the coronary artery, resulting

in damage from the suture rather than reﬂecting the intended

myocardial ischemia and infarction. Infarct sizes ⬍30% are

typically excluded. If included, small and large infarcts may

need to be grouped separately to reduce possible type II

statistical errors.

Cardiac wound healing and remodeling, typically assessed

days to weeks post-MI, can be examined using a wide variety

of approaches, including echocardiography, histology, bio-

chemistry, and cell biology (5, 217, 328). Serial measurements

of cardiac geometry and function by echocardiography are

useful for deﬁning phenotypes. Cardiac dimensions vary de-

pending on heart rate and depth of anesthesia, and these

parameters must be carefully controlled and matched across

groups. Cardiac functional reserve can be assessed by measur-

ing the contractile response to inotropic drugs or volume

overload. Cardiac MRI and hemodynamic assessment by pres-

sure-volume catheterization are other ways to measure cardiac

physiology parameters. It is feasible to quantify infarct size

noninvasively and serially by using cardiac MRI (181). Hemo-

dynamic evaluation in mice is a terminal procedure, which

prevents its use in serial assessments.

Hematoxylin and eosin staining provides information on

areas of necrosis and inﬂammation, while picrosirius red stain-

ing provides information on total collagen accumulation both

in the scar and remote regions (316). Immunohistochemistry

for neutrophils, macrophages, lymphocytes, ﬁbroblasts, and

endothelial cells provides information on the extent of inﬂam-

mation, scar formation, and neovascularization. Isolating indi-

vidual cell types and assessment of ex vivo phenotypes in

culture can further aid in understanding mechanisms. Studies

have revealed that inﬂammation evoked by acute myocardial

infarction also occurs systemically and that the spleen and liver

are important sources of cells and factors that inﬂuence LV

remodeling (71, 72, 95, 116, 198, 199, 293).

I/R MI: model rationale and variables measured. Implemen-

tation of myocardial reperfusion strategies has signiﬁcantly

reduced mortality in acute STEMI. Reperfusion has contrib-

uted to the growing pool of patients who survive the acute

event and are at risk for adverse remodeling and subsequent

development of heart failure (133, 136). In addition to salvag-

ing cardiomyocytes, reperfusion has profound effects on cel-

lular events responsible for repair and remodeling.

Although timely reperfusion is essential to salvage viable

cardiomyocytes from ischemic death, extensive preclinical and

clinical evidence suggests that reperfusion itself causes injury

(119, 121, 147). Reperfusion-induced arrhythmias and myo-

cardial stunning are self-limited and reversible forms of rep-

erfusion injury, while microvascular obstruction and lethal

cardiomyocyte injury are irreversible and extend damage, thus

contributing to adverse outcomes following MI (13, 126, 177,

241, 318). In patients, no reﬂow during reperfusion may be

exacerbated due to the generation of microemboli composed of

atherosclerotic debris and thrombi during percutaneous coro-

nary interventions (135, 253).

MI both with or without reperfusion shares many of the

same technical guidelines, and this information is provided

above. The one technical difference is whether the ligation is

removed at 45– 60 min after the occlusion to reperfuse the

myocardium. Similar to permanent occlusion MI, studies in-

vestigating the inﬂammatory and reparative response following

MI with reperfusion need to take into account the dynamic

sequence of cellular events involved in repair. Common mea-

surements shared by the two MI models are shown in Table 2.

For studies aimed at investigating acute myocardial injury

using a reperfusion strategy, the duration of coronary occlusion

needs to be sufﬁcient for the induction of signiﬁcant MI but not

overly prolonged to cause irreversible injury in the entire area

at risk. From the cell physiology perspective, the reparative

response after MI can be divided into three distinct but over-

lapping phases: inﬂammation, proliferation, and maturation

(26, 65). In the infarcted myocardium, dying cardiomyocytes

release damage-associated molecular patterns and induce cy-

tokines and chemokines to recruit leukocytes into the infarcted

region, thus triggering an intense inﬂammatory reaction that

serves to clear the infarct from dead cells and ECM debris,

while initiating a reparative response (84). Early reperfusion

after irreversible cardiomyocyte injury accelerates and accen-

tuates the inﬂammatory reaction and has profound effects on

the pathological features of the infarct. Microvascular hyper-

permeability is evident in the myocardium with acute I/R (97).

Rapid extravasation of blood cells through the hyperpermeable

vessels may result in hemorrhagic changes (98, 178). Inﬂux of

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phagocytotic macrophages is accelerated, resulting in more

rapid removal of dead cardiomyocytes compared with perma-

nent occlusion MI. In reperfused infarcts, dying cardiomyo-

cytes often exhibit large contraction bands, comprised of hy-

percontracted sarcomeres. Subsarcolemmal blebs and granular

mitochondrial densities, which are already present in irrevers-

ibly injured cardiomyocytes before restoration of blood ﬂow,

become more prominent upon reperfusion.

Phagocytosis of dead cells by activated macrophages results

in the activation of endogenous anti-inﬂammatory pathways,

ultimately leading to resolution of the inﬂammatory inﬁltrate.

Suppression of inﬂammation is followed by recruitment of

activated myoﬁbroblasts that deposit large amounts of ECM

proteins and by activation of angiogenesis (145). As the scar

matures, ﬁbroblasts become quiescent and infarct neovessels

acquire a coat of mural cells (332). Compared with large

mammals, rodents exhibit an accelerated time course of inﬁl-

tration with inﬂammatory and reparative cells (64).

Leukocyte inﬁltration during the inﬂammatory phase of

infarct healing and myoﬁbroblast activation and accumulation

during the proliferative phase are predominantly localized in

the infarct region and border zone (87, 122, 280). During scar

maturation, the cellular content in the infarcted region is

reduced. At the same time, however, the number of activated

macrophages and ﬁbroblasts in the remote remodeling myo-

cardium increases. Therefore, study of inﬂammatory and re-

parative cell inﬁltration and assessment of ECM protein depo-

sition should include systematic assessment of each end point

in the infarcted region, the peri-infarct area, and the remote

remodeling myocardium.

Sympathetic nerves are damaged by permanent coronary

occlusion but can regenerate after injury (220). In the setting of

chronic MI, regional hyperinnervation around the infarcted

region has been observed, and activation of cardiac sympa-

thetic nerves is important in triggering ventricular arrhythmias,

and such proarrhythmic action is dependent on the extent of

infarction (1, 70, 315, 326). In contrast, after I/R, chondroitin

sulfate proteoglycans prevent reinnervation (99, 100). Thus,

the model selected for sympathetic nerve evaluation should be

taken into consideration and depends on what question is being

asked.

MI: intervention considerations. The effects of interventions

on post-MI remodeling can be studied using both nonreper-

fused MI and reperfused MI/R models (11, 219, 232, 294,

322). Typically, nonreperfused MI yields accentuated dilative

remodeling and exacerbated dysfunction compared with a

reperfused infarct involving the same vascular territory, re-

ﬂecting a combination of more extensive infarct and less

effective repair. In the reperfused MI/R model, the effects of

genetic or pharmacologic interventions implemented early af-

ter reperfusion may reﬂect differences in the extent of acute

cardiomyocyte injury rather than differences in wound healing

responses. With permanent occlusion MI (assuming a stan-

dardized area at risk) or very late reperfusion models, differ-

ences in geometry and function of the remodeling heart are

independent of acute cardiomyocyte injury and reﬂect effects

on inﬂammatory, reparative, or ﬁbrotic cascades. In the pres-

ence of an occluded coronary artery, the delivery of systemi-

cally administered pharmacologic agents to the infarcted re-

gion of large animal models may be dependent on formation of

collaterals.

While the development of genetically targeted animals

(mice, rats, and rabbits) resulted in an explosion of studies

dissecting cell biological mechanisms and molecular pathways,

large animal models are considered closer to the clinical

situation for translational studies to test safety and effective-

Table 2. Common output measurements for in vivo MI and MI/reperfusion studies

Measurement Information Provided

Infarct size Infarct size (MI)

Infarct size per area at risk (MI/reperfusion)

Initial ischemic stimulus

Final area of damage

Effect of therapy or intervention

Plasma biomarkers Ischemia: creatine kinase, troponins

Inﬂammation: cytokines and chemokines

Scar formation: growth factors and the ECM

Neovascularization: angiogenic factors

Left ventricular physiology (echocardiography, MRI,

positron emission tomography imaging)

Geometry and function: dimensions, wall thickness, left ventricular dimensions and volumes,

fractional shortening, ejection fraction, remodeling index

Electrophysiological function: PR, QRS, and QT intervals/morphology; spontaneous and

inducible arrhythmias

Inﬂammation Immunohistochemistry and immunoblot analysis for cell numbers and inﬂammatory protein

expression

Flow cytometry analysis of the digested myocardium for individual cell phenotypes

Gene expression

Systemic and circulating inﬂammation

ECM scar Picrosirius red for collagen deposition

Immunohistochemistry and immunoblot analysis for ECM proteins and cross-linking enzymes

Gene expression

Scar strength assessment

Neovascularization Blood vessel numbers

Vessel type and quality

Microvascular damage Microvascular plugging

Hyperpermeability/edema

Hemorrhage

MI, myocardial infarction; ECM, extracellular matrix; MRI, magnetic resonance imaging.

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ness. Optimal study of molecular, cellular, and LV functional

end points and interpretation of the ﬁndings require under-

standing of the underlying pathophysiology. Assessment of

infarct size is typically the primary end point for investigations

examining the mechanisms of cardioprotection. Assessment of

chamber dimensions using echocardiography or MRI is crucial

to study the progression of adverse remodeling. Systolic and

diastolic cardiac geometry and function can be assessed non-

invasively using echocardiography (including Doppler ultra-

sound and speckle tracking), MRI, and hemodynamic assess-

ment. Mechanistic dissection of speciﬁc pathways may require

inclusion of additional cell physiology and molecular or pro-

teomic end points. In experimental models of MI, understand-

ing the time course of the cellular and molecular events is

critical for optimal study design. The effects of varying isch-

emic intervals on survival and activation of noncardiomyocyte

cellular and acellular (e.g., ECM) compartments are poorly

understood. Longer coronary occlusion times have distinct

effects on cardiac repair, by extending infarct size and by

inﬂuencing susceptible noncardiomyocyte populations, such as

endothelial cells, ﬁbroblasts, pericytes, and immune cells (85).

Most studies characterizing responses to myocardial injury

have so far been performed in healthy young animals. Comor-

bid conditions, such as aging, diabetes, and metabolic dysfunc-

tion, affect the pattern of ischemic injury and modify the time

course and qualitative characteristics of the inﬂammatory and

reparative responses (27, 106, 202, 238, 298, 319, 323). These

comorbidities are relevant in the clinical context and must be

considered in translation of experimental ﬁndings to the clinic.

To summarize, the major strengths and limitations of the

nonreperfused and reperfused MI models are shown in Table 1.

Ablation

Model rationale and variables measured. The primary ad-

vantages of ablative injury techniques such as cryo-, thermal-,

and radio-frequency ablation are rigid and reproducible control

over the size, shape, and location of the region of damage.

With such methods, a wound can be stamped on the target

myocardial tissue with consistent dimensions, shape, and trans-

mural depth. Because the size of the damaged region is inde-

pendent of animal-to-animal variations in coronary anatomy

(223), the resultant ablation scar is also more reproducible than

ligation-induced injury (52, 160, 307), aiding studies of long-

term structural and functional remodeling and providing better

power to detect the effects of an experimental drug or cell

therapy. Infarct location can thus be controlled independently

from coronary anatomy and infarct transmurality can be con-

trolled (52, 160, 290, 305, 307). There are, however, important

differences in the modes of cell death in ablative vs. occlusion

injuries. For example, cryoinjury results in necrosis due to the

generation of ice crystals and disruption of the cell membrane

rather than direct ischemia. Furthermore, ablative injuries are

typically generated from the epicardial surface inward,

whereas ischemic infarcts tend to be propagated outward from

the inner myocardial layers (52, 160).

Unlike MI or MI with reperfusion, cryoinjury kills all (or

nearly all) cells within the core of the damaged region and

creates distinct wound margins. Thus, a number of studies have

used cryoinjury to avoid confounding effects of resident sur-

viving cells when testing stem cell and other related therapies

(6, 7, 258, 302). Ablation procedures typically apply a cooled/

heated probe to either the epicardial or endocardial surface of

the heart. The extent and depth of the lesion depend on both the

temperature of the probe and the time it remains in contact with

the tissue; damage can be extended by generating multiple

adjacent lesions or by repeat application at the same location.

Because these physical factors are central to injury formation,

investigators should report probe size and material, tempera-

ture, method and duration of preheating/cooling, precise ana-

tomic location and duration of probe application, and interle-

sion time and number of lesions (if applicable). Cryoablation

has been used to generate reproducible wounds and scar tissue

for the study of myocardial injury response in various species

including dogs (160, 171, 297), rabbits (6, 7, 302), rats (49, 82,

149, 190), and mice (109, 204, 257, 289, 305, 307). In mice,

survival rate after cryoinjury was nearly twice that of perma-

nent coronary ligation over an 8-wk period (307), whereas

dysfunction was similar. Lower mortality may be a conse-

quence of smaller infarct size. Ablative methodologies have

also been used in nonmammalian species such as zebraﬁsh, to

probe the response of cardiac electrical properties to injury,

regeneration and scar formation (43, 46, 108). The ability to

destroy all cells within the cryoinfarct has provided interesting

clues regarding regeneration of fetal myocardium following

injury. In neonatal mice, mechanical or ischemic injuries to the

ventricular apex typically trigger regeneration, producing re-

covery of myocardial structure and function without scarring

(243, 276). Nontransmural cryoinfarcts in neonatal mice sim-

ilarly heal with minimal evidence of scarring and full func-

tional recovery with ongoing postnatal growth, while injuries

spanning the full thickness of the ventricular wall do not

regenerate muscle (57). Because neonatal mice can regenerate

myocardium during the ﬁrst postnatal week, models of myo-

cardial ischemia in neonatal mice may be used to identify

pathways involved in cardiac regeneration (8, 208).

As with coronary ligation models, evaluation of LV geom-

etry and function with echocardiography and assessment of

electrophysiological remodeling and arrhythmia risk are rou-

tinely performed in cryoinjury models. Given the early time

course and different mechanisms of necrotic injury in cryoin-

jury versus ligation models, cryoinjury studies are typically

more focused on long-term myocardial regeneration or me-

chanical/electrophysiological remodeling rather than mecha-

nisms of acute postinjury cell death, inﬂammation, and scar

formation (52, 160). Ablation procedures are now used com-

monly in clinical electrophysiology, and it is therefore not

surprising that a number of experimental electrophysiology

studies have taken advantage of geometric control provided by

this model (54, 231). Cryoinjury and ischemic injury differ in

their transmural localization and the amount of surviving

myocardium (52, 109, 257, 258, 305). Finally, methods for

ablative targeting of cardiac neural tissues have proven useful

in studies of the role of autonomic inputs in normal and

arrhythmic hearts (254).

To summarize, the major strengths and limitations of the

cryoinjury model are shown in Table 1.

Cardioprotection

Model rationale and variables measured. An intervention

that is cardioprotective is broadly deﬁned as serving to protect

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the heart (https://www.merriam-webster.com/dictionary/

cardioprotective), thereby in theory encompassing all of the

aforementioned aspects of cardiac damage and dysfunction. To

date, the only clinically established cardioprotective interven-

tion is early reperfusion (119, 133). The archetypal additive

cardioprotective intervention is, without question, ischemic

conditioning, encompassing the phenomena of ischemic pre-

conditioning, postconditioning, and remote conditioning (118,

175, 224, 247, 248, 329). Despite differences in the timing of

the protective stimulus (with preconditioning applied in a

prophylactic manner and postconditioning administered at the

time of reperfusion) and the site of the protective trigger (either

locally, or, for remote conditioning, in a tissue or organ distant

from the at-risk myocardium), all three forms of conditioning

share a common theme: there is overwhelming agreement that

ischemic preconditioning, postconditioning, and remote condi-

tioning render the heart resistant to lethal I/R-induced injury

(78, 118, 128, 163, 247).

This consensus with regard to ischemic conditioning and

cardioprotection is, however, a notable exception in the ﬁeld.

Indeed, for the vast majority of the innumerable cardioprotec-

tive strategies that have been investigated, the current preclin-

ical literature on the topic of cardioprotection is fraught with

controversies and a lack of reproducibility among investigators

and laboratories (163, 188). The ensuing confusion in the ﬁeld

may be attributed in part to two confounding factors: an overly

broad use of the term cardioprotection in some studies, to-

gether with false positive and false negative outcomes derived

from protocols executed in a suboptimal manner (162, 163,

188).

In some instances, a broad and suitably framed deﬁnition of

cardioprotection incorporating, for example, endothelial integ-

rity and vascular function, is appropriate (126, 225). A gener-

ally acknowledged and more narrowly focused hallmark of

cardioprotection is deﬁned as an agent or intervention that,

when administered in the setting of ischemia/reperfusion, aug-

ments myocardial salvage and reduces myocardial infarct size

beyond that achieved by reperfusion alone (128). Accordingly,

for our purposes, we focus on cardiomyocyte viability and

deﬁne cardioprotection as a strategy that attenuates cardiomy-

ocyte death. Cardiomyocyte viability can be assessed in a full

spectrum of models, ranging from cardiomyocytes in culture to

isolated buffer-perfused hearts to in vivo studies in rodents

(mice and rats) or larger animals (including rabbits, dogs, pigs,

sheep, and, in a small number of studies, primates). There is no

ideal model that completely mirrors the clinical scenario to

fully ensure absolute translational relevance. Rather, each

model has merits and disadvantages (as shown in Table 3).

The overwhelming strength of the mouse species is the

availability of genetically modiﬁed strains to elucidate molec-

ular mechanisms once candidate cardioprotective strategies

have been identiﬁed, a beneﬁt that is balanced by inherent

variability and resultant requirement for large sample sizes. An

additional problem of the mouse is the atypical geometry of

nontransmural infarcts, in which the subendocardium is spared

from death by diffusion of oxygen from the LV cavity and

occupies an inordinate proportion of the total LV wall thick-

ness. In all rodents, heart rate is much higher and therefore

infarct development is much faster than in larger mammals and

humans. Studies conducted in large animals (in particular, the

pig) are considered to have the greatest potential for preclinical

Table 3. Recommendations for cardioprotection studies

Model

Gold Standard

Primary End Point Required Covariates Potential Secondary End points Advantages Limitations

Cultured

cardiomyocytes

Cell viability (live-

dead assay)

None Types of cell death (i.e., apoptosis),

mitochondrial function; ROS

production

Capacity for high throughput Reductionist

Isolated buffer-perfused

hearts (mouse, rat,

and rabbit)

Infarct size (TTC) For models of regional

ischemia: area at

risk

Measures of LV function and coronary

ﬂow; cTn as a secondary index of

cardiac injury

Throughput higher than in vivo; eliminates

confounding effect of intervention on

systemic blood vessels

Reductionist

Mouse Infarct size (TTC) Area at risk;

hemodynamics

Measures of LV function; measures of

no reﬂow; CK or cTn as secondary

indexes of cardiac injury

Availability of genetically modiﬁed

strains; model of low collateral ﬂow

(measurement of RMBF not required)

Small size; differences between strains;

substantial variability requiring large

nvalues

Rat and rabbit Infarct size (TTC) Area at risk;

hemodynamics

Measures of LV function; measures of

no reﬂow; CK or cTn as secondary

index of cardiac injury

Reliable infarct production; relatively high

survival rate in experienced hands;

commercial availability of strains that

have comorbidities; model of low

collateral ﬂow (measurement of RMBF

not required)

Not high throughput

Dog Infarct size (TTC) Collateral blood ﬂow;

area at risk;

hemodynamics

Measures of LV function; measures of

no reﬂow; CK or cTn as secondary

indexes of cardiac injury

Large amount of historical data; shows

effect of intervention in the setting of

variable collateral ﬂow; mimics humans

with high collateral ﬂow

High cost; variability in collateral

perfusion (RMBF must be

measured); potential for lethal

arrhythmias; not high throughput

Pig Infarct size (TTC) Area at risk;

hemodynamics

Measures of LV function; measures of

no reﬂow; CK or cTn as secondary

indexes of cardiac injury

Model of low collateral ﬂow

(measurement of RMBF not required);

mimics humans with low collateral ﬂow

High cost; high incidence of lethal

arrhythmias; not high throughput

TTC, triphenyltetrazolium chloride; LV, left ventricular; CK, creatine kinase; cTn, cardiac troponin; RMBF, regional myocardial blood ﬂow.

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relevance (103, 140). This advantage is accompanied by sub-

stantial costs incurred and, particularly in pigs, the well-

documented high incidence of lethal ventricular arrhythmias

(282).

In all studies focused on cardioprotection, the primary end

point must be a quantitative assessment of cardiomyocyte

viability. In cell culture models, these data may be obtained

using a live-dead assay such as trypan blue exclusion, pro-

pidium iodide exclusion, or other commercially available as-

says. In intact hearts, including isolated buffer-perfused hearts

and all in vivo models, the gold standard end point is myocar-

dial infarct size by TTC staining and, at later time points,

histopathologic analysis (79, 81). For models involving reper-

fusion, the area at risk must be quantiﬁed and infarct size must

be expressed as a proportion of the risk region. With global

rather than regional ischemia/reperfusion, the entire heart is

rendered at risk and thus infarct size is appropriately expressed

as a proportion of the total ventricular area. The duration of

ischemia must be sufﬁcient to cause signiﬁcant infarction but

not complete death of at-risk cardiomyocytes in the control

cohort. That is, if the duration of ischemia is selected such that

infarct size in controls is either inordinately small or exces-

sively large, the scope for salvage and probability of achieving

cardioprotection with any intervention is negligible. Irrespec-

tive of the model used, the protocol must involve I/R (or, in cell

culture models, H/R) rather than ischemia (or hypoxia) alone.

This requirement reﬂects the fact that even the most powerful

and well-established cardioprotective strategies such as isch-

emic preconditioning simply delay, rather than prevent, the

progression to cardiomyocyte death and infarction (224, 324).

The duration of reperfusion must be sufﬁcient to allow for the

accurate and unambiguous delineation of necrotic and viable

myocardium. This is of particular importance when infarct size

is quantiﬁed using TTC staining: a minimum of 1–2 h of

reperfusion is considered mandatory in rodent hearts, while

longer periods of at least 3 h are standard in large animal

models (283).

Attention must be paid to essential covariates and possible

cofounders. Important considerations for all in vivo models

include body temperature, the choice of anesthetics and anal-

gesics, and changes in the determinants of myocardial oxygen

supply and demand, all of which are well recognized to have

profound effects on infarct size (111, 247). Particular care must

be taken to avoid the possibility of inadvertent preconditioning:

examples include triggering a protective phenotype by unin-

tentionally subjecting the myocardium to brief periods of

hypoxia or ischemia during surgical preparation, or intention-

ally imposing a period of ischemia in an effort to identify the

extent of the at-risk myocardium (180). Additional covariates

and confounders are model speciﬁc. In rodents, age, sex, and

strain of the animals have all been implicated or identiﬁed to

inﬂuence myocardial infarct size (10, 111, 306) Circadian

variation, the time of day at which experiments are performed,

may also be important (16, 28, 110, 266). The canine model is

known for its variability in the magnitude of collateral blood

ﬂow. Accordingly, when using this model, measurement of

regional myocardial blood ﬂow during coronary artery occlu-

sion and incorporation of collateral ﬂow as a covariate in the

analysis of infarct size are mandatory (64, 284).

Finally, the overwhelming majority of studies conducted to

date have assessed the efﬁcacy of candidate cardioprotective

strategies using healthy, juvenile, or adult animals. There is

evidence that the infarct-sparing effect of these purportedly

protective interventions may be lost or attenuated in the setting

of clinically relevant comorbidities, including aging, type 1 and

type 2 diabetes, hypercholesterolemia, and hypertension, and

may be inﬂuenced by diet or exercise (3, 4, 51, 78, 115, 117,

146, 150, 186, 197, 246, 249, 321). Accordingly, once proof of

principle is established, it is imperative that promising cardio-

protective therapies be reevaluated, adhering to the essential

elements of rigor described above, in comorbid models (127).

All protocols must include concurrent and appropriate control

cohorts. For example, when potential cardioprotective drugs

are evaluated, controls must receive matched volumes of ve-

hicle administered in an identical manner.

THE ISSUE OF TRANSLATION: TOWARD A RANDOMIZED

CONTROLLED STUDY OR TRIAL DESIGN

There is currently no established intervention, aside from

timely reperfusion, that limits damage to hearts of patients

experiencing myocardial ischemia to the extent that clinical

outcome is improved (133, 138). Discussions of prior failures

and hope for future successes have been reviewed elsewhere

(120, 127, 176). Several elements missing from preclinical

studies of infarct size reduction have been identiﬁed and

include absence of critical investigator blinding, statistical

weaknesses (underpowered studies), and insufﬁcient method-

ological detail. These deﬁciencies explain in part the failure to

translate preclinical results into effective infarct-sparing treat-

ments in patients. Thus, many have questioned the reproduc-

ibility of interventions to protect from MI (i.e., reduce infarct

size).

Much of the lack of reproducibility has been ascribed to

limited or lacking scientiﬁc rigor (29, 250). In response to these

and other concerns, the United States National Institutes of

Health now includes explicit requirements for applicants to

show, and reviewers to evaluate, the level of scientiﬁc rigor in

grant applications. Whether suboptimal rigor fully explains and

underlies the reproducibility crisis is unclear (162). Nonethe-

less, advocating for reproducibility and scientiﬁc rigor is wel-

come.

Appropriate statistical issues should be considered before

initiating an infarct-sparing intervention. Investigators should

know the standard deviation of their primary, prespeciﬁed end

point, such as infarct size, chamber dimension, or ejection

fraction. A power analysis for the primary end point will

determine and justify the number of subjects to be enrolled in

each group. This may not be feasible when investigating an

entirely innovative strategy as there may be no basis for an

estimation of the expected effect size. The standard deviation

of the investigator’s most recent blinded study can be used to

determine group size (229). The choice of statistical analyses

must be appropriate for the study design (300). For two-group

studies, t-tests (or nonparametric equivalent) may be used; for

protocols involving multiple cohorts, ANOVA (or nonpara-

metric equivalent) is mandatory. Analysis of covariance, as-

sessing the effects including variations in risk region and

collateral blood ﬂow, may also be applied.

Blinded assignment of animals and randomization to control

or treated groups is mandatory whenever possible. Incorpora-

tion of randomization with blinding is an easy and logical

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approach. For testing classical drug-based interventions or

when comparing mutant mice, individuals responsible for

blinding can use block randomization and label tubes or mice

with a simple unique alphanumeric code. The surgeon per-

forming the protocol should have no knowledge of the inter-

vention or genotype. We recommend that the same surgeon

perform all surgeries; if multiple surgeons are used, equal

numbers from all groups should be matched across the surgeon

pool. It is imperative that neither the individual analyzing

infarct size, nor the individual performing any other secondary

analyses, knows the intervention or genotype until all data are

compiled. If the potential for excluding animals exists, this

should be done based on previously declared exclusion and

inclusion criteria, and all decisions should be made before

disclosure of group assignment; details of such exclusions

should be made clear in any publications. Table 4 shows

ARRIVE guidelines for manuscript submission, modiﬁed to

focus on ischemia studies.

The Consortium for Preclinical Assessment of Cardiopro-

tective Therapies (CAESAR) endeavored to address the pri-

mary issues related to reproducibility in cardioprotection stud-

ies (163). Performing the same protocol at multiple sites and in

multiple species was extraordinarily challenging; the most

notable were challenges in identifying the underlying explana-

tions for differences in infarct size (or other variables) between

centers while they were implementing consonant protocols.

Interestingly, there were several instances of mice being or-

dered simultaneously from the same vendor, only to have

signiﬁcantly different body weights at the time of study (de-

spite being fed the same chow). To be clear, the weight

differences were relatively small but on occasion were signif-

icantly different for reasons unknown. It is likely that small

differences, such as this, could theoretically affect the results

(or the perception of not being able to reproduce studies) of

published studies. During the CAESAR experience, numerous

seemingly extraneous factors were considered, such as differ-

ences in municipal water source, room temperature, relative

humidity, type of lighting, trafﬁc in the room, and other

seemingly innocuous details that may or may not affect the

responsiveness of mice to an infarct-sparing regimen. All of

these variables reﬂect inherent challenges in performing in

vivo studies.

More important than slight differences in body weight or

other such ancillary factors were initial challenges in generat-

ing equivalent infarct sizes at different institutions, despite

using the same protocol. Such occasional variations emphasize

the unequivocal requirement in all protocols for concurrent and

appropriate controls (see Cardioprotection).

OVERALL DISCUSSION AND CONCLUSIONS

As highlighted throughout these guidelines, ischemia and

I/R have multiple consequences that show temporal variation

in terms of both incidence and inﬂuence on outcomes and may

be model dependent. Examples range from acute effects (in-

cluding biochemical perturbations in cardiomyocytes and other

cardiac cell types, disruption in cardiac conduction and devel-

opment of arrhythmias, contractile dysfunction, abnormalities

in endothelial, and vascular reactivity) to longer term outcomes

(such as cardiomyocyte death, microvascular obstruction and

no-reﬂow, scar healing, and LV remodeling) and, ultimately,

major adverse cardiac events, including heart failure and death

(136). Figure 1 shows the diversity in models available to

assess ischemia across its spectrum, and Tables 4 and 5 show

general recommendations for rigor and reproducibility in isch-

emia studies.

While writing these guidelines, the authors discussed

whether an algorithm to deﬁne the choice of model for a given

scientiﬁc question would be helpful. The consensus was that

the topic of myocardial ischemia and infarction and the many

clinical manifestations of coronary artery disease resulting in

and from myocardial ischemia or infarction are so broad and so

complex that we ﬁnd ourselves unable to provide an algorithm

that truly covers all potential scientiﬁc approaches. Indeed,

such an algorithm may be used by regulatory and funding

agencies to actually limit research in the ﬁeld, which would be

counterproductive to the goals of this document.

The approach used will vary depending on the questions

being addressed; as such, all of the approaches described above

may be considered good and a gold standard if appropriate to

address the target hypothesis. In vitro studies using isolated

cardiomyocytes or even isolated organelles (e.g., mitochon-

dria) are well suited to identify single molecular targets of

injury and protection (102). Isolated, buffer-perfused heart

Table 4. Checklist of considerations for rigor and reproducibility, modiﬁed from the ARRIVE Guidelines (168)

Item Details

Ethical statements Institutional Animal Care and Use Committee approval, Guide for the Care and Use of Laboratory Animals, welfare

assessments and interventions

Animal description, housing,

husbandry

Species, strain, source, age (mean and range), sex, genotypes, body weight (mean and range), health/immune status, housing

type (speciﬁc pathogen free), cage type, bedding material, number of cage companions, light-dark cycle, room

temperature and humidity, food type, food and water access

Study design Deﬁne model used; deﬁne groups; matching, randomization, and blinding protocols; order of treatment and assessment of

groups; method to conﬁrm model success; inclusion/exclusion criteria (e.g., lower limit for infarct size); daily monitor to

record time and cause of death; deﬁne timing of perioperative and postoperative phases for survival analysis, ﬂow chart

for complex designs

Experimental procedures Deﬁne area examined (e.g., infarct, remote, both regions); positive and negative controls; for drugs: formulation, dose, site,

and route of administration; anesthesia and analgesic use and pain monitoring; surgical procedure details and monitoring

records (e.g., electrocardiogram, heart rate, and anesthesia depth); method of euthanasia; time of day performed

Sample sizes Number of animals used per group for each experiment; sample size calculation; number of independent replicates for cell

culture studies

Variables measured Primary and secondary end points, list any animals or samples removed from analysis with reason

Statistics Methods used for each analysis; test for assumptions

ARRIVE, Animals in Research: Reporting In Vivo Experiments.

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models can be used to study the acute biochemical and func-

tional mechanisms of myocardial I/R injury and cardioprotec-

tion. Due to the need for stable stenosis and the required spatial

resolution of regional myocardial blood ﬂow and contractile

function measurements, large animal preparations are recom-

mended as models for the clinical manifestations of chronic

stable angina and coronary microembolization. In vivo prepa-

rations are required to study more long-term effects of myo-

cardial I/R and respective therapeutic interventions. We distin-

guish between permanent occlusion MI and reperfused MI

models and also highlight measurements in common. Perma-

nent coronary occlusion MI and reperfused MI models are both

well suited to study repair and remodeling. I/R models are

mandatory to study cardioprotection, and the ischemia must be

of sufﬁcient severity and duration to cause some infarction.

Cryo-/thermoinjury is not suited to study the pathophysiology

of MI but well suited to study repair and regeneration pro-

cesses.

Prospective planning of study design, i.e., randomization for

appropriate control versus treatment, blinding of investigators

(as much as possible for a given experimental protocol and the

subsequent data analysis), and adequate statistics, is mandatory

for reproducibility of all experimental models of myocardial

I/R and infarction. As larger data sets are being acquired,

consideration for how to harness big data should be given (272,

274). Compiling databases to incorporate results from across

studies and across laboratories will provide a means to use

epidemiological approaches or big data tools to validate pub-

lished ﬁndings, generate novel hypotheses, and assess individ-

ual variability in response to ischemia.

In conclusion, these guidelines provide recommendations to

help the investigator plan and execute a full range of studies

involving myocardial ischemia and infarction.

GRANTS

We acknowledge support from the following: National Institutes of

Health Grants HL-002066, HL-051971, HL-056728, HL-061610, HL-

075360, HL-078825, HL-088533, HL-092141, HL-093579, HL-107153,

HL-111600, HL-112730, HL-112831, HL-113452, HL-113530, HL-

116449, HL-128135, HL-129120, HL-129823, HL-130266, HL-131647,

HL-132075, HL-135772, GM-103492, HL-131647, HL-76246, HL-85440,

GM-104357, GM-114833, GM-115428, and UL1-TR-001412; American

Heart Association Grants 16GRNT30960054 and 16CSA28880004; Grand

Challenge Award; Department of Defense Grants 16W81XWH-16 –1-0592,

PR151051, PR151134, and PR151029; Biomedical Laboratory Research and

Development Service of the Veterans Affairs Ofﬁce of Research and Devel-

opment Awards 1IO1BX002659 and I01BX000505; Australian National

Health and Medical Research Council Research Fellowship APP1043026;

Bundesministerium für Bildung und Forschung Grant BMBF01 EO1004; and

German Research Foundation Grants DFG He 1320/18-3 and SFB 1116 B8.

R. A. Kloner reports grant support from Stealth Biotherapeutics, Servier, Inc.,

and Faraday to test experimental compounds in experimental myocardial

infarction models.

DISCLAIMERS

The content is solely the responsibility of the authors and does not

necessarily represent the ofﬁcial views of the National Institutes of Health,

American Heart Association, United States Department of Defense, United

States Veterans Administration, National Health and Medical Research Coun-

cil, or German Research Foundation.

Table 5. Recommendations for ischemia studies

Common Experimental design should follow ARRIVE guidelines (see Table 4)

Cardiomyocytes When comparing groups, geometry and function end points by echocardiography assessment may not change. This

does not necessarily indicate no effect of the intervention.

Control groups can be shared across studies to reduce animal use, as long as the samples are collected within the

same timeframe, under identical conditions, and details are provided in the methods. Previously collected historical

controls should be avoided or clearly indicated.

For time-course studies, sham surgery can be replaced by day 0 negative controls if minimally invasive procedures

are used, which greatly reduces animal use.

Use to discern direct cardiomyocyte effects and responses

Isolated perfused hearts Use to discern cardiac effects and responses

Angina, stunning, hibernation, and

ischemic cardiomyopathy Use to reﬂect a particular clinical scenario

MI Use nonreperfused or reperfused MI to study repair and remodeling

Use nonreperfused MI to test interventions in a robust remodeling model and to test interventions in a model

clinically relevant to the nonreperfused patient

Use reperfused MI to test interventions in a model clinically relevant to the reperfused patient

Use reperfused MI to study cardioprotection

Essential to measure infarct size for nonreperfused MI and infarct size and area at risk for reperfused MI

Ablation Use to control size, shape, or location

Use to achieve maximal and uniform cell death in the target region

Use to investigate mechanisms of action of corresponding to clinical ablation technique

While not suited to study MI pathophysiology, is well suited to study repair and regeneration

Essential to quantify transmural extent of damage, to assess transmural variation

Essential to recognize that transmural extent of the lesion may evolve over time

Essential to standardize experimental protocol (e.g., probe temperature and contact time) to achieve consistent lesions

Cardioprotection Use to evaluate potentially protective strategies in the ischemia-reperfusion model

Essential to measure infarct size and area at risk

ARRIVE, Animals in Research: Reporting In Vivo Experiments; MI, myocardial infarction.

H828 MYOCARDIAL ISCHEMIA GUIDELINES

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DISCLOSURES

No conﬂicts of interest, ﬁnancial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.L.L. and G.H. conceived and designed research; M.L.L., R.G.G., K.P.,

C.M.R., and G.H. prepared ﬁgures; M.L.L., R.B., J.M.C., X.-J.D., N.G.F., S.F.,

R.G.G., J.W.H., S.P.J., R.K., D.J.L., R.L., E.M., P.P., K.P., F.A.R., L.S.L.,

C.M.R., J.E.V.E., and G.H. drafted manuscript; M.L.L., R.B., J.M.C., X.-J.D.,

N.G.F., S.F., R.G.G., J.W.H., S.P.J., R.K., D.J.L., R.L., E.M., P.P., K.P.,

F.A.R., L.S.L., C.M.R., J.E.V.E., and G.H. edited and revised manuscript;

M.L.L., R.B., J.M.C., X.-J.D., N.G.F., S.F., R.G.G., J.W.H., S.P.J., R.K.,

D.J.L., R.L., E.M., P.P., K.P., F.A.R., L.S.L., C.M.R., J.E.V.E., and G.H.

approved ﬁnal version of manuscript.

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Visualization of intracellular ATP dynamics in the whole kidney under pathophysiological conditions using the kidney slice culture system

Preprint

May 2024

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated ATP synthesis pathway, and analyzed intracellular ATP dynamics using ex vivo IR and cisplatin nephropathy models. We demonstrated that proximal tubules (PTs) were strongly dependent on oxidative phosphorylation (OXPHOS), whereas podocytes relied on both OXPHOS and glycolysis using ATP synthesis inhibitors. We also confirmed that the ex vivo IR model could recapitulate ATP dynamics in vivo ; ATP recovery in PTs after reperfusion varied depending on ischemia length, whereas ATP in distal tubules (DTs) recovered well even after long ischemia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent delayed cisplatin-induced ATP decrease in PTs. This novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

Numerical Modeling of Myocardial Infarction in Multivessel Coronary Lesion. I. Analysis of Some Model Scenarios

Article

Jun 2024

The study of the inflammatory phase of acute myocardial infarction in multivessel coronary lesion was performed using the methodology of mathematical modeling. The minimal reaction-diffusion mathematical model is focused on the description of the functional M1/M2 polarization of macrophages and the influence of factors of aseptic inflammation on the process of cardiomyocyte death. The initial conditions and dynamics of the process in the infarction nucleus are assumed to be consistent with laboratory measurement data. The nature of the spatiotemporal distribution of substances (cell populations and inflammatory mediators) and the features of the formation of nonlinear dynamic structures of demarcation inflammation are studied using model examples. The patterns of functioning of the basic mechanisms of the inflammatory response are analyzed, and the role of the main inflammatory mediators is evaluated. The previously obtained estimates of the effectiveness of anti-inflammatory therapeutic strategies based on cytokine management and macrophage polarization in complex heart attack scenarios with multivessel coronary lesion have been confirmed. The research results allow us to consider the accepted reaction-diffusion model with constant diffusion coefficients as an example of a formal mathematical description of an active environment in which dissipative (diffusion) and local biochemical processes compete with each other, as well as the pro-inflammatory link of innate immunity opposes the anti-inflammatory one. The ability of macrophages to functionally M1/M2 polarization and reprogramming plays a crucial role in this competition.The adequacy of the research results is confirmed by quantitative and qualitative agreement with experimental data.

Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart

Preprint

Jun 2024

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

Pharmacological inhibition of RUNX1 reduces infarct size after acute myocardial infarction in rats and underlying mechanism revealed by proteomics implicates repressed cathepsin levels

Article

Full-text available

Jun 2024
FUNCT INTEGR GENOMIC

Myocardial infarction (MI) results in prolonged ischemia and the subsequent cell death leads to heart failure which is linked to increased deaths or hospitalizations. New therapeutic targets are urgently needed to prevent cell death and reduce infarct size among patients with MI. Runt-related transcription factor-1 (RUNX1) is a master-regulator transcription factor intensively studied in the hematopoietic field. Recent evidence showed that RUNX1 has a critical role in cardiomyocytes post-MI. The increased RUNX1 expression in the border zone of the infarct heart contributes to decreased cardiac contractile function and can be therapeutically targeted to protect against adverse cardiac remodelling. This study sought to investigate whether pharmacological inhibition of RUNX1 function has an impact on infarct size following MI. In this work we demonstrate that inhibiting RUNX1 with a small molecule inhibitor (Ro5-3335) reduces infarct size in an in vivo rat model of acute MI. Proteomics study using data-independent acquisition method identified increased cathepsin levels in the border zone myocardium following MI, whereas heart samples treated by RUNX1 inhibitor present decreased cathepsin levels. Cathepsins are lysosomal proteases which have been shown to orchestrate multiple cell death pathways. Our data illustrate that inhibition of RUNX1 leads to reduced infarct size which is associated with the suppression of cathepsin expression. This study demonstrates that pharmacologically antagonizing RUNX1 reduces infarct size in a rat model of acute MI and unveils a link between RUNX1 and cathepsin-mediated cell death, suggesting that RUNX1 is a novel therapeutic target that could be exploited clinically to limit infarct size after an acute MI.

Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury

Article

Full-text available

May 2024
BASIC RES CARDIOL

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2−/− → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2−/−vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

Evaluation of Vasodilatory Effect of Nitroglycerin in Cardioplegia Solution on Patients Undergoing Coronary Artery Bypass Graft Surgery

Article

Full-text available

May 2023

Cardioprotection in cardiovascular surgery

Article

Full-text available

Jun 2024
BASIC RES CARDIOL

Since the invention of cardiopulmonary bypass, cardioprotective strategies have been investigated to mitigate ischemic injury to the heart during aortic cross-clamping and reperfusion injury with cross-clamp release. With advances in cardiac surgical and percutaneous techniques and post-operative management strategies including mechanical circulatory support, cardiac surgeons are able to operate on more complex patients. Therefore, there is a growing need for improved cardioprotective strategies to optimize outcomes in these patients. This review provides an overview of the basic principles of cardioprotection in the setting of cardiac surgery, including mechanisms of cardiac injury in the context of cardiopulmonary bypass, followed by a discussion of the specific approaches to optimizing cardioprotection in cardiac surgery, including refinements in cardiopulmonary bypass and cardioplegia, ischemic conditioning, use of specific anesthetic and pharmaceutical agents, and novel mechanical circulatory support technologies. Finally, translational strategies that investigate cardioprotection in the setting of cardiac surgery will be reviewed, with a focus on promising research in the areas of cell-based and gene therapy. Advances in this area will help cardiologists and cardiac surgeons mitigate myocardial ischemic injury, improve functional post-operative recovery, and optimize clinical outcomes in patients undergoing cardiac surgery.

CircNSD1 promotes cardiac fibrosis through targeting the miR-429-3p/SULF1/Wnt/β-catenin signaling pathway

Article

May 2024

Guidelines for Assessing Maternal Cardiovascular Physiology During Pregnancy and Postpartum

Article

May 2024
AM J PHYSIOL-HEART C

Maternal mortality rates are at an all-time high across the world and are set to increase in subsequent years. Cardiovascular disease is the leading cause of death during pregnancy and postpartum, especially in the US. Therefore, understanding the physiological changes in the cardiovascular system during normal pregnancy is necessary to understand disease-related pathology. Significant systemic and cardiovascular physiological changes occur during pregnancy that are essential for supporting the maternal-fetal dyad. The physiological impact of pregnancy on the cardiovascular system has been examined in both experimental animal models and in humans. However, there is a continued need in this field of study to provide increased rigor and reproducibility. Therefore, these guidelines aim to provide information regarding best practices and recommendations to accurately and rigorously measure cardiovascular physiology during normal and cardiovascular-disease-complicated pregnancies in human and animal models.

Activation of the non-neuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury

Article

May 2024
AM J PHYSIOL-HEART C

The intracardiac nervous system is considered to be involved in ischemic conditioning’s cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC’s protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

A Novel Method of Standardized Myocardial Infarction in Aged Rabbits

Article

Full-text available

Feb 2017
AM J PHYSIOL-HEART C

Guidelines for Measuring Cardiac Physiology in Mice

Article

Full-text available

Jan 2018

Cardiovascular disease is a leading cause of death, and translational research is needed to understand better mechanisms whereby the left ventricle responds to injury. Mouse models of heart disease have provided valuable insights into mechanisms that occur during cardiac aging and in response to a variety of pathologies. The assessment of cardiovascular physiological responses to injury or insult is an important and necessary component of this research. With increasing consideration for rigor and reproducibility, the goal of this guidelines review is to provide best-practice information regarding how to measure accurately cardiac physiology in animal models. In this article, we define guidelines for the measurement of cardiac physiology in mice, as the most commonly used animal model in cardiovascular research. Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/guidelines-for-measuring-cardiac-physiology-in-mice/.

A canine model of chronic ischemic cardiomyopathy: characterization of regional flow-function relations

Article

Full-text available

Feb 1999

The controversy regarding the mechanism(s) of left ventricular (LV) dysfunction in chronic coronary artery disease is, in part, related to the lack of an appropriate animal model for this condition. We have developed such a model by placing Ameroid constrictors on proximal portions of coronary arteries in dogs who were euthanized (mean of 6 wk) after the development of severe global LV dysfunction noted on two-dimensional echocardiography. The LV end-systolic size nearly doubled ( P < 0.001) over the observation period, and the percent change in LV size from end diastole to end systole decreased by >50% ( P< 0.001). Regional dysfunction was noted in 23 of 24 myocardial beds analyzed within regions showing no gross evidence of infarction. In 10 of these beds, severe dysfunction was noted without a decrease in radiolabeled microsphere-derived myocardial blood flow (MBF). In 13 myocardial beds, decrease in function was associated with a decrease in MBF ( P < 0.001), with close coupling noted between percent wall thickening and MBF. In the beds that exhibited an ultimate decrease in MBF, the decrease in function preceded the decrease in MBF. In conclusion, we describe chronic LV dysfunction in a canine model of multivessel stenosis that closely mimics chronic ischemic LV dysfunction in humans. Whereas regional function is severely reduced in this model, MBF is varied in different segments and at different times during the observation period. These results provide new insights regarding flow-function relations in chronic ischemic LV dysfunction.

STAT3 as a common signal of ischemic conditioning: a lesson on “rigor and reproducibility” in preclinical studies on cardioprotection

Article

Full-text available

Nov 2017
BASIC RES CARDIOL

Ischemic conditioning before (ischemic preconditioning, IPC) or after (ischemic postconditioning, POCO) sustained myocardial ischemia/reperfusion (I/R), induced locally or remotely from the heart (remote IPC, RIPC), reduces infarct size. However, none of the identified signaling steps of ischemic conditioning was robust across models and species to be successfully translated to humans. In prior separate studies in pigs, activation of signal transducer and activator of transcription 3 (STAT3) was causal for infarct size reduction by IPC, POCO, and RIPC but it remains unclear whether or not STAT3 is truly a common denominator of cardioprotective signaling. We therefore, now analyzed the phosphorylation of STAT3 and other signaling proteins in left ventricular biopsies from our prior studies on IPC, POCO and RIPC in one approach. We developed a strategy for the quantification of protein phosphorylation in multiple samples from many experiments on different gels/membranes by Western blot. Along with reduced infarct size, the ratio of STAT3tyr705 phosphorylation/total STAT3 protein at early reperfusion was significantly increased by IPC (IPC 2.0 ± 0.3 vs. I/R 1.2 ± 0.2 arbitrary units), but only trendwise by POCO and RIPC (1.3 ± 0.2; 1.4 ± 0.2 arbitrary units); storage time for IPC samples was shorter than for POCO and RIPC samples. No other signaling protein phosphorylation was associated with reduced infarct size. We confirmed STAT3 phosphorylation with IPC. For POCO and RIPC we could not reproduce the findings from our earlier more focused studies. At this point, we can not distinguish between lack of robustness of the biological signal and methodological issues of our retrospective approach.

Heart Functional and Structural Compendium of Cardiosplenic and Cardiorenal Networks in Acute and Chronic Heart Failure Pathology

Article

Full-text available

Nov 2017
AM J PHYSIOL-HEART C

Heart failure (HF) secondary to myocardial infarction (MI) is linked to kidney complications that comprise cellular, structural, functional, and survival indicators. However, HF research is focused on left ventricular (LV) pathology. Here, we determined comprehensive functional analysis of the LV using echocardiography in transition from acute heart failure (AHF) to progressive chronic heart failure (CHF) pathology and developed a histological compendium of the cardiosplenic and cardiorenal networks in pathological remodeling. In surgically induced MI using permanent coronary ligation, the LV dysfunction is pronounced, with myocardium necrosis, wall thinning, and 20–30% LV rupture events that indicated AHF and CHF pathological remodeling in C57BL/6 male mice (2–4 mo old, n = 50). Temporal LV function analysis indicated that fractional shortening and strain are reduced from day 1 to day 5 in AHF and sustained to advance to CHF from day 28 to day 56 compared with naïve control mice ( n = 6). During the transition of AHF ( day 1 to day 5) to advanced CHF ( day 28 to day 56), histological and cellular changes in the spleen were definite, with bimodal inflammatory responses in kidney inflammatory biomarkers. Likewise, there was a unidirectional, progressive, and irreversible deposition of compact collagen in the LV along with dynamic changes in the cardiosplenic and cardiorenal networks post-MI. The renal histology and injury markers suggested that cardiac injury triggers irreversible dysregulation that actively alters the cardiosplenic and cardiorenal networks. In summary, the novel strategies or pathways that modulate comprehensive cardiosplenic and cardiorenal networks in AHF and CHF would be effective approaches to study either cardiac repair or cardiac pathology. NEW & NOTEWORTHY The present compendium shows irreversible ventricular dysfunction as assessed by temporal echocardiography while histological and structural measurements of the spleen and kidney added a novel direction to study cardiosplenic and cardiorenal networks in heart failure pathology. Therefore, the consideration of systems biology and integrative approach is essential to develop novel treatments. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/temporal-dynamics-of-acute-and-chronic-heart-failure/ .

VEGF Nanoparticles Repair Heart after Myocardial Infarction

Article

Full-text available

Nov 2017

Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine that has been shown to promote neovascularization in hearts of patients with ischemic heart disease but can also lead to adverse effects depending on the dose and mode of delivery. We investigated whether prolonged exposure to a low dose of VEGF could be achieved by encapsulating VEGF in polylactic coglycolic acid nanoparticles and whether treatment with VEGF-containing nanoparticles improved cardiac function and protected against left ventricular remodeling in the hearts of mice with experimentally induced myocardial infarction. Polylactic coglycolic acid nanoparticles with a mean diameter of ~113 nm were generated via double emulsion and loaded with VEGF; the encapsulation efficiency was 53.5 ± 1.7% (107.1 ± 3.3 ng VEGF/mg nanoparticles). In culture, VEGF nanoparticles released VEGF continuously for at least 31 days, and in a murine myocardial infarction model, VEGF nanoparticle administration was associated with significantly greater vascular density in the peri-infarct region, reductions in infarct size, and improvements in left ventricular contractile function 4 wk after treatment. Thus, our study provides proof of principle that nanoparticle-mediated delivery increases the angiogenic and therapeutic potency of VEGF for the treatment of ischemic heart disease. NEW & NOTEWORTHY Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine but has a short half-life and a rapid clearance rate. When encapsulated in nanoparticles, VEGF was released for 31 days and improved left ventricular function in infarcted mouse hearts. These observations indicate that our new platform increases the therapeutic potency of VEGF.

Proteomics/phosphoproteomics of left ventricular biopsies from patients with surgical coronary revascularization and pigs with coronary occlusion/reperfusion: Remote ischemic preconditioning

Article

Full-text available

Dec 2017

Remote ischemic preconditioning (RIPC) by repeated brief cycles of limb ischemia/reperfusion reduces myocardial ischemia/reperfusion injury. In left ventricular (LV) biopsies from patients undergoing coronary artery bypass grafting (CABG), only the activation of signal transducer and activator of transcription 5 was associated with RIPC’s cardioprotection. We have now used an unbiased, non-hypothesis-driven proteomics and phosphoproteomics approach to analyze LV biopsies from patients undergoing CABG and from pigs undergoing coronary occlusion/reperfusion without (sham) and with RIPC. False discovery rate-based statistics identified a higher prostaglandin reductase 2 expression at early reperfusion with RIPC than with sham in patients. In pigs, the phosphorylation of 116 proteins was different between baseline and early reperfusion with RIPC and/or with sham. The identified proteins were not identical for patients and pigs, but in-silico pathway analysis of proteins with ≥2-fold higher expression/phosphorylation at early reperfusion with RIPC in comparison to sham revealed a relation to mitochondria and cytoskeleton in both species. Apart from limitations of the proteomics analysis per se, the small cohorts, the sampling/sample processing and the number of uncharacterized/unverifiable porcine proteins may have contributed to this largely unsatisfactory result.

Molecular and Cellular Mechanisms of Myocardial Stunning

Article

Apr 1999

The past two decades have witnessed an explosive growth of knowledge regarding postischemic myocardial dysfunction or myocardial “stunning.” The purpose of this review is to summarize current information regarding the pathophysiology and pathogenesis of this phenomenon. Myocardial stunning should not be regarded as a single entity but rather as a “syndrome” that has been observed in a wide variety of experimental settings, which include the following: 1) stunning after a single, completely reversible episode of regional ischemia in vivo; 2) stunning after multiple, completely reversible episodes of regional ischemia in vivo; 3) stunning after a partly reversible episode of regional ischemia in vivo (subendocardial infarction); 4) stunning after global ischemia in vitro; 5) stunning after global ischemia in vivo; and 6) stunning after exercise-induced ischemia (high-flow ischemia). Whether these settings share a common mechanism is unknown. Although the pathogenesis of myocardial stunning has not been definitively established, the two major hypotheses are that it is caused by the generation of oxygen-derived free radicals (oxyradical hypothesis) and by a transient calcium overload (calcium hypothesis) on reperfusion. The final lesion responsible for the contractile depression appears to be a decreased responsiveness of contractile filaments to calcium. Recent evidence suggests that calcium overload may activate calpains, resulting in selective proteolysis of myofibrils; the time required for resynthesis of damaged proteins would explain in part the delayed recovery of function in stunned myocardium. The oxyradical and calcium hypotheses are not mutually exclusive and are likely to represent different facets of the same pathophysiological cascade. For example, increased free radical formation could cause cellular calcium overload, which would damage the contractile apparatus of the myocytes. Free radical generation could also directly alter contractile filaments in a manner that renders them less responsive to calcium (e.g., oxidation of critical thiol groups). However, it remains unknown whether oxyradicals play a role in all forms of stunning and whether the calcium hypothesis is applicable to stunning in vivo. Nevertheless, it is clear that the lesion responsible for myocardial stunning occurs, at least in part, after reperfusion so that this contractile dysfunction can be viewed, in part, as a form of “reperfusion injury.” An important implication of the phenomenon of myocardial stunning is that so-called chronic hibernation may in fact be the result of repetitive episodes of stunning, which have a cumulative effect and cause protracted postischemic dysfunction. A better understanding of myocardial stunning will expand our knowledge of the pathophysiology of myocardial ischemia and provide a rationale for developing new therapeutic strategies designed to prevent postischemic dysfunction in patients.

Transient denervation of viable myocardium after myocardial infarction does not alter arrhythmia susceptibility

Article

Nov 2017
AM J PHYSIOL-HEART C

Cardiac sympathetic nerves stimulate heart rate and force of contraction. Myocardial infarction (MI) leads to the loss of sympathetic nerves within the heart, and clinical studies indicate that sympathetic denervation is a risk factor for arrhythmias and cardiac arrest. Two distinct types of denervation have been identified in the mouse heart after MI caused by ischemia-reperfusion: transient denervation of peri-infarct myocardium, and sustained denervation of the infarct. Sustained denervation is linked to increased arrhythmia risk, but it is not known if acute nerve loss in peri-infarct myocardium also contributes to arrhythmia risk. Peri-infarct sympathetic denervation requires the p75 neurotrophin receptor (p75NTR), but removal of p75NTR alters the pattern of sympathetic innervation in the heart and increases spontaneous arrhythmias. Therefore, we targeted the p75NTR co-receptor sortilin, and the p75NTR-induced protease TACE/ADAM17 (Tumor necrosis factor-α Converting Enzyme/A disintegrin and metalloproteinase domain 17) in order to selectively block peri-infarct denervation. Sympathetic nerve density was quantified using immunohistochemistry for tyrosine hydroxylase. Genetic deletion of sortilin had no effect on the timing or extent of axon degeneration, but inhibiting TACE/ADAM17 with the protease inhibitor marimastat prevented the loss of axons from viable myocardium. We then asked if retention of nerves in peri-infarct myocardium had an impact on cardiac electrophysiology three days after MI, using ex vivo optical mapping of transmembrane potential and intracellular calcium. Preventing acute denervation of viable myocardium after MI did not significantly alter cardiac electrophysiology or calcium handling, suggesting that transient denervation at this early timepoint has minimal impact on arrhythmia risk.

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing following myocardial infarction

Article

Oct 2017

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after MI. Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages mice show better outcomes post-MI, while aged TG mice show a worse aging phenotype, we wanted to evaluate the combination effect to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17-28 month old male and female C57BL/6J wild type (WT) mice and TG mice (n = 10-21/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day (D) 7 post-MI, TG aging mice showed improved diastolic properties and remodeling index compared to WT (both P<0.05). Macrophage numbers were higher in TG compared to WT at both D0 and D7 post-MI, and the post-MI increase was due to elevated CD18 protein levels (all P<0.05). RNA-seq analysis of cardiac macrophages isolated from D7 post-MI infarcts identified 1,276 statistically different genes (994 increased and 282 decreased in TG, all P<0.05). Reduced vascular endothelial growth factor-A (VEGFA), platelet-derived growth factor subunit A (Pdgfa), and transforming growth factor beta 3 (Tgfb3) along with elevated tissue inhibitor of MMP-4 (Timp4) macrophage expression reveal mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG compared to WT at both D0 and D7 post-MI (P<0.05 for both), the post-MI collagen cross-linking ratio was higher in the WT (P<0.05), consistent with increased diastolic volumes. Vessel numbers (by GSL-I lectin staining) were decreased in the TG compared to WT at both D0 and D7 post-MI (P<0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling.

Guidelines for Experimental Models of Myocardial Ischemia and Infarction

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