Genetic Modification of the Heart
Murine physiology: measuring the phenotype

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Abstract

The phenotype is the observable and measurable characteristic (or biochemical trait) of an organism that results from the interaction of the environment with the expression of gene alleles (i.e. the genotype). Accurate phenotyping demands that findings are not missed, anticipated, or biased; a mixture of assays that characterize distinct, but interrelated aspects of the cardiovascular phenotype are often necessary to meet these requirements. Such stringent conditions are crucial because it is phenotypic analysis that ultimately determines the utility of genetically altered and spontaneous mutant mice for biomedical research.

Section snippets

Gene-based and phenotype-based strategies

While gene targeting and transgenesis have provided important insights into the molecular and cellular mechanisms that underlie cardiovascular development, function, and disease, a major limitation of these gene-based approaches is the need to identify a candidate gene that is presumptively involved in the pathogenesis of a disease state. However, most traits and diseases are genetically complex, resulting from combinations of several genes (polygenic and epistatic traits) or from interactions

Conceptual approaches to phenotypic analysis

The approach to cardiovascular phenotypic analysis can be arbitrarily categorized as hierarchical, ontogenetic, or pyramidal (Table 1). The hierarchical method employs complementary studies at the molecular, myocyte, muscle, isolated (Langendorff or working) heart and intact organism levels; the success of this approach is based on the premise that studies at several levels ultimately yield a clearer picture of the impact of the transgenic modification than a study at a single level. The

The in vivo murine phenotype

Due to its small size and rapid heart rate (HR), functional analysis of the cardiac phenotype in the transgenic mouse remains challenging; fortunately, miniaturization and refinement of techniques used to study the cardiovascular system in larger animals have largely met this challenge. A major concern relates to the availability and cost (equipment acquisition and trained staff to operate the instruments) of accessing these new technologies [7].

Using the techniques described in this review,

Exercise and the phenotype

Exercise is a highly integrated physiologic stress commonly used to evaluate cardiovascular disease, and is an ideal stress to uncover pathologic alterations in cardiovascular reserve. The cardiovascular response to exercise has been characterized in mice [15] and exercise and metabolic studies have been performed in a variety of genetically engineered mouse models [16], [17], [18]. The use of exercise in transgenic mouse models has been recently reviewed [19]. Examples include mice lacking β1-

Echocardiography

Two-dimensionally directed M-mode echocardiography is unquestionably the leading method for imaging the cardiovascular system in small animals. It is non-invasive, versatile, readily available, and well suited for serial studies over a wide range of ages. M-mode echocardiography has been used to phenotype the cardiovascular system in a variety of genetic mouse models.

Current ultrasonographs employ linear array broadband transducers with small footprints that operate at 12–15 MHz. These

Pressure

Accurate and reproducible measurements of systolic BP can be obtained in conscious mice with tail cuff plethysmography [69], [70]. Pressure corresponding to the systolic BP is externally applied to the tail in the restrained, conscious animal and is measured by a manometer attached to a tail cuff; a photoresistor detects blood flow in the tail. Computerized systems that perform all functions automatically have been validated against intra-arterial BP measurement [69], [70]. An important

Flow

Cineangiography and microspheres have been used to measure flow [82], [83], but have significant limitations and currently, are uncommonly performed. X-ray contrast microangiography was used to quantitate RV dilation and dysfunction and tricuspid regurgitation in mice with chronic pulmonary arterial constriction [58]. Aortic flow can be measured with aortic flow probes in the acutely instrumented animal and provide a “gold” reference standard [37], [43], [52]. Cardiac index determinations were

Bioelectricity

Single-lead ECG is used to time and gate events (e.g. echo or MRI), and multiple-lead ECG is used for cardiac diagnosis in the anesthetized animal [99]. Telemetry devices can be implanted for long-term analysis of arrhythmias [100] and HR variability (HRV) [101] in the conscious mouse. More detailed, sophisticated phenotypic analysis is possible with electrophysiologic (EP) studies.

Selecting an in vivo assay

Several issues should be considered when selecting an in vivo assay (Table 3). First, accuracy, reproducibility and diagnostic discrimination of the assay may not be similar among laboratories, particularly with a technique that has been investigated by only a few groups. Regional and national resource centers have databases of selected phenotypic assays with careful quality control and offer a potential solution, but creating and maintaining searchable databases of cardiovascular phenotypes

Conclusions

It is clear from this brief survey that each of the methods used to study integrated cardiovascular physiology in the adult mouse has its advantages and limitations. Accordingly, it is imperative that the investigator selects studies that are tailored to the specific hypotheses and questions being addressed. In most instances, a combination of ex vivo and in vivo techniques are required to understand the physiologic significance of a genetic perturbation and in many cases, complimentary

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