First identified in 2000, ACE2 is known to be highly expressed in human heart and may act to oppose the activity of the renin-angiotensin system by converting Ang II to the vasodilator Ang I-7 and by competing with ACE for Ang I [
1,
2,
4,
10]. In addition, recent animal data suggest that ACE2 can modulate cardiac contractility, suppress cardiac Ang II levels and down-regulate hypoxia-induced pro-apoptotic genes [
8], pointing to a role for ACE2-derived peptides in the regulation of cardiac function. Given that overactivity of the renin-angiotensin system is implicated in the pathophysiology of chronic heart failure, this study explored whether cardiac ACE2 may be differentially regulated in this condition. The technique of QRT-PCR was applied as we have found this to be the most quantitative and sensitive technique for probing changes in the expression of specific genes in human tissues [
11].
To validate the use of QRT-PCR to identify differentially regulated genes in heart failure, ANF was studied as a positive control. As previously reported [
12], ANF mRNA was found to be abundantly expressed in left ventricular myocardium and to be up-regulated in idiopathic dilated cardiomyopathy. ANF mRNA expression was also 8-fold up-regulated in the ischemic cardiomyopathy group although the change (relative to control) narrowly missed being statistically significant (P = 0.058). Several components of the renin-angiotensin system were subsequently studied. In agreement with previous workers [
13], ACE mRNA was found to be up-regulated in both forms of heart failure, in accordance with the hypothesis that local production of Ang II is enhanced in the failing heart. Interestingly, ACE2 appeared more highly expressed than ACE in the non-failing ventricle and, like ACE, the levels of ACE2 mRNA were found to be significantly increased in idiopathic and ischemic cardiomyopathy.
The observation that ACE2 appears more highly expressed than ACE in human left ventricle should be interpreted cautiously as differences in primer probe set efficiencies can make difficult the quantitative comparison of expression levels of different targets. In this case, primer probe sets for ACE and ACE2 are believed to be of similar efficiency since they gave similar threshold values when used in a quantitative PCR with 10,000 genomic equivalents as the template. Therefore, the current data coupled to previous reports [
3,
14] suggests that the relative expression levels of ACE:ACE2 mRNA is likely to vary significantly dependent on the species and tissue.
Unlike mRNA expression for ACE and ACE2, that of the angiotensin AT
1 receptor was not found to be differentially expressed in failing myocardium. This contrasts with previous data [
15,
16] indicating reduced AT
1receptor expression in heart failure. Since the latter studies were carried out on right ventricular biopsies, it is possible that regional variation occurs in ventricular AT
1 receptor expression in the failing heart. Finally, in contrast to all other genes studied, no expression (zero copies) of renin mRNA was detected in either non-failing or failing left ventricular myocardium. The question of whether renin may be produced locally in the myocardium has been an issue of controversy over the years but has not to our knowledge previously been addressed using QRT-PCR. However, even with this sensitive detection methodology, no ventricular expression (zero copies) was detected. This was not a methodology failure as the primer probe set used for renin transcript amplification worked as expected in human renal cortex. Therefore, these data are in agreement with previous findings [
17], suggesting that the renin gene is not expressed in cardiac tissues.
Therefore, the key novel result from the current work is that ACE2 mRNA expression is increased in the human failing heart. This finding sits well with the emerging hypothesis that ACE2 may counterbalance the effects of stimulation of the renin-angiotensin system by reducing Ang II levels, and generating peptide products such as Ang1-7, which may benefit cardiac function. Indeed, recent data suggest that administration of Ang 1-7 can ameliorate the development of ischemia-induced heart failure in rats [
18,
19]. Since the completion of the current study, it has been discovered that the G protein coupled receptor, Mas, is a high affinity, functional receptor for Ang 1-7 [
20]. The question of whether Mas, like ACE2, is also differentially regulated in heart failure will be an interesting subject for future studies to address. Whether up regulation of ACE2 has important functional consequences in the human failing heart remains to be determined, however, the emerging data raise the intriguing possibility that a novel therapeutic approach to heart failure may be achieved through enhancement of ACE2 activity.
It is worth noting some limitations of the current work. First, only changes in mRNA have been addressed. While in the case of ACE and ANF it has been shown by other workers that in the failing heart increased mRNA translates into increased protein, this remains to be proven for ACE2. In addition, it will be of interest to determine which cell types in the failing heart increase the expression of ACE2 mRNA and protein. Secondly, while the experimental groups were matched for sex and age as closely as possible, the donors in both the heart failure groups were receiving various pharmacological therapies. Thus, we must acknowledge the possibility that these therapies could have influenced the levels of expression of ACE2 mRNA. Clarification of this question will likely require studies in animal models of heart failure where background can be strictly controlled. Finally, only end stage heart failure has been examined in this study, thereby giving no temporal data regarding the expression of ACE2 during the development of failure.
1 Martin J Goddard,2 Jennifer C Allen,1 and Kenneth L Clark1
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