Cardiomyocyte-specific Prolyl-4-hydroxylase Domain 2 Knock Out Protects from Acute Myocardial Ischemic Injury

Abstract

Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the ␣-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd ؊/؊ ) When oxygen availability is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level (1). Hypoxia-inducible factors (HIFs) 2 are essential players in these responses because they regulate the transcription of a large number of genes that affect a myriad of cellular processes, including angiogenesis, metabolism, cell survival, and oxygen delivery (2). HIF is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit HIF-1␣ or the more cell type-specifically expressed HIF-2␣ or HIF-3␣ and the oxygen-insensitive ␤-subunit (3). In the presence of oxygen, HIF␣ becomes hydroxylated at two critical proline residues by prolylhydroxylase domain (PHD) enzymes (4, 5). The PHD protein family responsible for HIF␣ regulation consists of three members called prolylhydroxylase domain (PHD)1, PHD2, and PHD3 (6, 7). Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein recognizes ⌯⌱F-1␣ subunits and targets them for rapid ubiquitination and proteasomal degradation (8 -10).Based on the ubiquitous expression pattern and its dominant effect in normoxia, it had to be assumed that PHD2 is the most critical HIF-1␣-regulating PHD isoform in most tissues (11)(12)(13). This notion, learned from in vitro studies, was confirmed by the up to now available genetically modified Phd2 mouse models (14). Phd2 knock-out embryos die between embryonic day (E) 12.5 and E14.5 (15). This time point coincides with the increased levels of PHD2 in wild-type (wt) mice starting from E9.0. A major role of PHD2 in regulating the HIF system is further underscored by mouse models with a somatic Phd2 Ϫ/Ϫ knock out, which enable to analyze the in vivo function of PHD2 in the adult mice. Two independent inducible Phd2 Ϫ/Ϫ mouse models were developed by Takeda et al. (16) and Minamishima et al. (17). The phenotype of these mice most obviously resembles the consequences of HIF␣ overexpression with increased angiogenesis, erythropoiesis, and extramedullar hematopoiesis (17,18). Most interestingly, these mice also develop a cardiac phenotype with symptoms of dilated cardiomyopathy. In the heart, HIF-1␣ and thereby also the PHDs are known to influence key components of heart development, morphogenesis, and function (19,20). Long term activation of HIF-1␣ in the heart seems to activate detrimental pathways resulting in the development of heart failure (21). Thus, it is tempting to speculate that loss of PHD2 in the heart is responsible for the dilated cardiomyopathy as observed in the inducible Phd2 Ϫ/Ϫ mice. However, because these mice also develop an inc...

Preischemic targeting of HIF prolyl hydroxylation inhibits fibrosis associated with acute kidney injury

“…In addition to timing, the duration of PHD inhibition/HIF activation appears to play a critical role in determining disease outcome. Mice with cardiomyocyte-specific Phd2 deletion, for example, had significantly improved cardiac function 3 wk after myocardial IRI (15), whereas sustained Phd2 ablation or tissue-specific HIF-1␣ stabilization resulted in cardiomyopathy (22). While we previously showed that epithelial cell-derived HIF-1 functions as a profibrotic transcription factor in experimental models of CKD (12,13), transient global pre-or postischemic HIF stabilization did not produce adverse effects.…”

PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban

“…When oxygen availability is limited, PHD enzymatic activity will be inhibited, resulting in stabilization of HIF-1α protein and activation of the HIF-1 pathway (10). Therefore, it is not surprising that pretreatment with prolyl hydroxylase inhibitor or depletion of PHD2, the major prolyl hydroxylator of HIF-α, activates the HIF pathway and elicits HIF-mediated adaptive responses before the onset of hypoxia, attenuating cardiac injury induced by subsequent myocardial ischemia (16)(17)(18)(19). However, it was recently reported that long-term depletion of both PHD2 and PHD3 in the heart resulted in myocardial dysfunction and produced many hallmarks of ischemic cardiomyopathy (23).…”

“…Marked induction of HIF-α protein was observed in the region close to the infarct area in a rat model of MI (16). Interestingly, pretreatment with prolyl hydroxylase inhibitor or depletion of PHD2 or PHD3 attenuates myocardial injury induced by myocardial ischemia in several rodent models (16)(17)(18)(19)(20). It was suggested that the HIF pathway plays a central role in this cardioprotective effect (18,21).…”

Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition: Robust New Target or Another Big Bust for Stroke Therapeutics?

“…RNA interference showed that deletion of HIF PHD1 but not deletion of HIF PHD2 or HIF PHD3 can provide protection against oxidative death of neurons (Siddiq et al, 2009). Conditional deletion of all HIF PHDs has been shown to affect cell viability in response to developmental stimuli in sympathetic neurons (HIF PHD3; Bishop et al, 2008), ischemia in muscle (HIF PHD1; Aragones et al, 2008), and ischemia in the heart (HIF PHD2; Holscher et al, 2011), but these studies while promising, seem to have limited import for posttreatment protection. Future studies using a combination of conditional knockouts and specific pharmacological inhibitors will clarify whether isoform selective HIF PHD inhibition is a viable treatment strategy after stroke.…”