Cardioprotective effect of St. Thomas’ Hospital No. 2 solution against age-related changes in aquaporin-7-deficient mice

Aquaporins (AQPs) are transmembrane proteins that regulate transcellular water flow. Mammalian AQPs are present in many tissues and organs, such as the kidneys, brain, lungs, digestive system, eyes, and skin [1]. AQPs are involved in diverse physiological and pathophysiological processes, particularly in cerebral ischemia, congestive heart failure, hypertension, and angiogenesis. Nevertheless, many effects of cardiac AQPs are yet to be elucidated [1]. To date, 13 AQPs have been identified in mammals (AQP0–12, encoded by the genes AQP0–AQP12): they are divided into three groups: the classical group (AQP1, 2, 4, and 5) that selectively transfers water; the aqua-glycero-porin group (AQP3, 7, 9, and 10) that transfers both water and small molecules (e.g., glycerol); and the unorthodox group that includes un-categorizable AQP6, 8, 11, and 12 [2]. Several AQP subtypes have been discovered in the cardiac tissues of various animals and humans using real-time polymerase chain reaction [3‐5].

Cardiomyocytes require fatty acids and glucose for energy production [6, 7]. A recent study showed that glycerol was an important cardiac energy substrate and that AQP7 was a glycerol facilitator [8]. AQP7 deficiency increases myocardial infarction size and apoptosis after ischemia [9]. AQP7 expression is downregulated in obese patients [6]. Analysis of the AQP7 sequence in 160 human subjects revealed three missense mutations, one of which was the AQP7-G264V protein with impaired glycerol and water transport functions [10]. We previously reported the relationship between AQP7 and myocardial injury against normothermic global ischemia (GI) and elucidated the efficacy of St. Thomas’ Hospital No. 2 solution (STH2), a hyperkalemic cardioplegia, in a mature murine heart with AQP7 deficiency [11]. Unfortunately, AQP7-KO mice exhibited obesity and insulin resistance after 12 weeks [12]. Further investigations comparing the effects of STH2 on ischemia–reperfusion injury among different age groups are needed. To better understand these associations, the present study aimed to investigate whether STH2 was equally effective in young and aged AQP7-KO mice and to elucidate the mechanisms by which intra-myocardial adenosine triphosphate (ATP) content was altered during ischemia in AQP7-KO mice.

The present study investigated the relationship between AQP7 and myocardial injury against normothermic GI and elucidated the efficacy of hyperkalemic cardioplegia in young and aged murine hearts with AQP7 deficiency. We found that changes in intra-myocardial ATP content in AQP7-KO mice were comparable to those in WT mice in the Langendorff perfused system. These findings indicate that AQP7 deficiency does not influence the myocardial protective effect of STH2 in hyperkalemia depolarized cardioplegia.

AQP7 is a membrane protein that is a member of the glycerol and water molecule-transporting aqua-glycero-porin family. Accordingly, AQP7 is also involved in the passive transfer of other small uncharged solutes, such as urea, purines, arsenite, ammonia, and hydrogen peroxide [14]. AQP7 deficiency has also been shown to result in insulin resistance [15]. Some AQPs have been recently found to function as ion channels, and the effects of divalent cations on the ionic conductance of AQPs have been studied. Kourghi et al. reported that human AQP1-expressing oocytes showed little effect of Mg²⁺, in contrast to moderate inhibition by Ca²⁺ [16]. Given that STH2 contains magnesium chloride, which inhibits Ca²⁺ channels and provides additional cardio-protective effects, it is an important clinical question to understand whether Mg²⁺ affects the regulatory function of AQP7.

AQP7 is highly expressed in white and adipose tissue. Meanwhile, AQP7 is weakly expressed in the heart, skeletal muscle, and kidneys. Glycerol produced by the hydrolysis of triglycerides in adipocytes is efficiently released by AQP7 [17]. Méndez-Giménez et al. [18] found increased AQP7 expression in the adipose tissues of experimental rats with diet-induced obesity undergoing bariatric surgery. Another mouse analysis showed that AQP7 mRNA levels in the quadriceps femoris muscles were significantly higher in mice with diet-induced obesity than in control chow-fed mice [19]. Immuno-histochemical analysis of the muscles of mice with diet-induced obesity showed enhanced expression of AQP7 at the myofiber surface membranes, suggesting that AQP7 facilitated the secretion of glycerol from myocytes. A similar phenomenon was observed in another study using obese leptin-deficient ob/ob mice [20].

Wakayama et al. [21] demonstrated that AQP7 mRNA expression in the myocardium, as well as in the skeletal muscle, was two times higher in db/db mice than in WT mice. Interestingly, Hibuse et al. [12] found no difference in body weight between young WT and AQP7-KO, although AQP7-KO mice developed obesity and insulin resistance at age 12 weeks due to increased glycerol kinase (Gyk) activity [12]. In the present study, no remarkable difference was found between the body weight of young WT and AQP7-KO mice (28.8 ± 1.9 g vs 27.7 ± 2.2 g). Meanwhile, in aged mice, although the average body weight was still not significantly different (39.2 ± 6.7 g in WT mice vs 42.5 ± 2.1 g in AQP7-KO mice), there was smaller inter-individual variability in KO mice than in WT mice. A recent clinical study on subcutaneous abdominal fat biopsies reported that AQP7 expression in adipose tissue was lower in obese subjects than in lean subjects [22]. The gene expression of AQP7 in different animal species and organs during obesity should be further investigated.

Hibuse et al. [8] reported that myocardial glycerol content under normal feeding conditions was significantly lower in AQP7-KO mice than in WT mice. Furthermore, the cardiac ATP content was significantly lower in AQP7-KO mice than in WT mice [8]. They assessed energy metabolism to examine the consumption of energy substrates using isolated murine hearts perfused in the Langendorff mode and found no differences in palmitic acid or glucose consumption, whereas the glycerol consumption rate was significantly reduced in AQP7-KO mice. The intracellular ATP content increased with the addition of glucose, even under AQP7 knockdown. However, the increase in intracellular ATP content induced by the addition of glycerol was significantly blocked in AQP7-KO mice. The authors found that glycerol acted as an energy substrate in the heart via AQP7.

Ischemic conditions activate glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3 phosphate to dihydroxyacetone phosphate to facilitate ATP synthesis from glycerol. The increase in glycerol-mediated ATP production under hypoxic conditions is suppressed by a GPD2 inhibitor in a dose-dependent manner [9]. In addition, Ishihama et al. [9] reported that AQP7 deficiency in mice increased myocardial infarct size after left coronary artery ligation and myocardial infarct-induced cardiac apoptosis in response to ischemia. Moreover, atrial natriuretic peptide levels in the heart, which increase upon cardiac dysfunction, were elevated 7 days after ligation in AQP7-KO mice [9]. In the present study, hearts were rapidly arrested with STH2, and the intracellular ATP content was significantly more preserved than in untreated hearts. As we used KHB-containing glucose rather than glycerol, further investigation is required using KHB with glycerol.

The hearts were perfused with standard KHB, including glucose, without glycerol or palmitic acid, and thus, further studies are warranted to assess metabolic adaptation and nutrition-related conditions (i.e., a specific diet or starvation). Myocardial ischemic disease is a multifactorial process involving a spectrum of injuries that affect myocardial protection. However, the hearts used in this experimental study were obtained from healthy WT and AQP7-KO mice under normal feeding conditions. It is likely that any protective effect of STH2 cardioplegia might be different in hearts with ischemic injury, hypertrophy, or in older hearts. In addition, damaged hearts are likely to require prolonged periods of ischemia to correct a lesion; however, the ischemic duration adopted in this experimental study is relatively short. Hence, it is necessary to investigate the use of longer ischemic durations and repeated administration of cardioplegia in accordance with clinical conditions. Ischemic tolerance and AQP function may be altered under hypothermic conditions in normal clinical practice.

The present study reveals the cardio-protective efficacy of STH2 in an experimental model of isolated AQP7-KO young and aged (> 6 months) murine hearts and the preservation of intra-myocardial ATP during ischemia by STH2. The findings suggest the safety of this cardiac-protective strategy even when the AQP7 gene is downregulated due to obesity or missense mutations.