The Pitfalls of in vivo Cardiac Physiology in Genetically Modified Mice - Lessons Learnt the Hard Way in the Creatine Kinase System

Abstract

In order to fully understand gene function, at some point, it is necessary to study the effects in an intact organism. The creation of the first knockout mouse in the late 1980's gave rise to a revolution in the field of integrative physiology that continues to this day. There are many complex choices when selecting a strategy for genetic modification, some of which will be touched on in this review, but the principal focus is to highlight the potential problems and pitfalls arising from the interpretation of in vivo cardiac phenotypes. As an exemplar, we will scrutinize the field of cardiac energetics and the attempts to understand the role of the creatine kinase (CK) energy buffering and transport system in the intact organism. This story highlights the confounding effects of genetic background, sex, and age, as well as the difficulties in interpreting knockout models in light of promiscuous proteins and metabolic redundancy. It will consider the dose-dependent effects and unintended consequences of transgene overexpression, and the need for experimental rigour in the context of in vivo phenotyping techniques. It is intended that this review will not only bring clarity to the field of cardiac energetics, but also aid the non-expert in evaluating and critically assessing data arising from in vivo genetic modification.

Keywords: creatine kinase; heart failure; integrative physiology; metabolism; transgenic.

Figure 1

Creatine biosynthesis and the myocardial creatine kinase (CK) phosphagen system. Arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1) is predominantly expressed in the kidneys where it combines arginine and glycine to make guanidinoacetate (GAA) or with lysine to make homoarginine (hArg). Circulating GAA is taken up by the liver where GAA N-methyltransferase (GAMT; EC 2.1.1.2) utilises the methyl group from S-adenosyl L-methionine to synthesise creatine. These biosynthetic enzymes are not expressed in cardiomyocytes so creatine must be taken-up via a specific plasma membrane creatine transporter (SLC6A8). Creatine accumulates inside the cell where the sarcomeric mitochondrial isoform of creatine kinase (MtCK; EC:2.7.3.2) catalyses the transfer of a phosphoryl group from ATP to form phosphocreatine (PCr) and ADP. PCr is a high abundance and mobile energy source that can be utilised to rapidly regenerate ATP at times of high demand under the control of the muscle isoform of creatine kinase (the dimer MMCK; EC 2.7.3.2). Free creatine diffuses back to stimulate further oxidative phosphorylation and re-start the cycle. Knockout models exist for all five of these proteins and overexpression models for CK and creatine transporter (CrT). This figure was created using Servier Medical Art by Servier, which is licensed under a Creative Commons Attribution 3.0 Unported License https://smart.servier.com.

Figure 2

The presence or absence of cardiac hypertrophy in CK double knockout mice (CK-dKO) has varied considerably over time and between laboratories, in large part, due to a mixed and changing genetic background. When finally bred onto a pure C57BL/6 J background, no cardiac hypertrophy was evident (Lygate et al., 2012a, - pure).