In this study, we investigated the role and mechanism of Kir6.1 in DCM. We found that the cardiac function and Kir6.1 expression were decreased in DCM mice. Kir6.1 overexpression improved cardiac dysfunction and upregulated the phosphorylation of AKT and Foxo1 in DCM, both in vivo and in vitro. In contrast, cardiac-specific Kir6.1 knockout aggravated the cardiac dysfunction and downregulated the phosphorylation of AKT and Foxo1 in DCM mice. Activation of Foxo1 downregulated the expression of Kir6.1 and decreased the ΔΨm in cardiomyocytes. In contrast, inactivation of Foxo1 upregulated the expression of Kir6.1 and increased the ΔΨm in cardiomyocytes. Furthermore, Foxo1 was shown to interact with the promoter region of Kir6.1 for transcription activation.
Diabetic cardiomyopathy is becoming a well-known clinical phenomenon. The metabolic milieu associated with diabetes, such as hyperglycemia and hyperinsulinemia, alters multiple molecular pathways within the cardiomyocyte, thereby impairing cardiac contractility and promoting myocyte dysfunction, injury, and cell death [1]. Systolic and diastolic dysfunction can be consistently reproduced in a variety of rodent models of diabetes. Echocardiography is a standard modality for diagnosing DCM. There are three myocardial signals in DCM: left ventricular (LV) diastolic dysfunction, abnormal LV systolic function, and changes in LV geometry [14]. Alteration in the BNP level suggests myocardial structural and functional dysfunction. Elevated BNP levels showed a positive correlation with LV dysfunction in DCM [15]. Cardiomyocyte hypertrophy is a common structural hallmark in patients with DCM [16, 17]. A relatively oxygen-poor environment induced by hypertrophy accelerates cardiomyocyte apoptosis. Cardiac function is energetically demanding, and thus reliant on efficient well-coupled mitochondria to generate adenosine triphosphate (ATP). Extensive experimental results demonstrated that cardiomyocytes from animal models of type 1 and 2 diabetes had altered mitochondrial morphology and mitochondrial dysfunction [18].
The mitoKATP subunit, Kir6.1, plays a major role in maintaining mitochondrial function. Alteration in mitochondrial function has been linked to cardiovascular diseases including DCM [19, 20]. Furthermore, numerous studies have shown the cardioprotective roles of mitoKATP [21]. In our study, the expression of Kir6.1 was decreased in the mouse model of type 2 DCM. In accordance with the in vivo results, it was decreased in a cardiomyocyte model of insulin resistance, which was also consistent with a previous study [9]. The data indicated that Kir6.1 may play a special role in DCM. Therefore, transgenic mice overexpressing Kir6.1 or lacking Kir6.1 specifically in the heart were used to study the role of Kir6.1 in DCM. Previous studies have shown that AAV-9 leads to preferential cardiac transduction in vivo [11, 22]. Moreover, cardiomyocytes can be efficiently transfected by adenoviruses. Our data showed that Kir6.1 expression was overexpressed in vivo and in vitro after AAV-9 or adenoviral gene transfer, respectively. The ability to control the tissue specificity of gene knockout in the rodent using the Cre-loxP technology has profoundly advanced rodent genetics and the ability to examine single gene functions in vivo [23]. We used the Cre-loxP technology to modify gene expression in our mouse model. The expression of Kir6.1 in the heart was significantly decreased after intraperitoneal tamoxifen injection, indicating that the cardiac-specific Kir6.1-knockout mouse model was successfully established.
In this study, we found that the cardiac function in DCM mice was decreased, including systolic and diastolic dysfunction, increase in BNP, cardiomyocyte hypertrophy and apoptosis, and abnormal changes in mitochondrial structure in vivo. Additionally, we found increased BNP levels and reduction in the OCR in vitro. DCM and its associated mitochondrial dysfunction have been observed in ob/ob, db/db and HFD-fed mice [24, 25]. Furthermore, cardiac tissue from Akita mice displayed swollen mitochondria, lacking a well-defined cristae structure along with decreased states 3 and 4 respiration and ATP synthesis [26]. Our data agree with many previous studies on cardiac dysfunction in rodent models of DCM [27–31]. However, in the current study, Kir6.1 overexpression reduced cardiac dysfunction in diabetic mice and dysfunction of cardiomyocytes with insulin resistance, whereas cardiac-specific Kir6.1 knockout aggravated cardiac dysfunction in diabetic mice. Thus, our findings suggest that Kir6.1 overexpression attenuates cardiac dysfunction in DCM.
Cardiac insulin signaling mediates cellular homeostasis by controlling substrate use, protein synthesis, autophagy, and cell survival [32]. Physiologically, binding of insulin to insulin receptor (IR) activates insulin receptor substrate 1 and 2 (IRS1 and IRS2) and the downstream phosphoinositide 3-kinase (PI3K)-AKT pathways. AKT is required for cardiac growth, metabolism, and survival, and its targets include p70S6K (protein synthesis), Glut4 (glucose transport), and Foxo1 (gene expression) [33]. Briefly, insulin exerts its function through AKT activation, which in turn phosphorylates Foxo1. In cardiomyocytes, Foxo1 is involved in the control of many important properties such as cell growth, metabolic adaptation, cell apoptosis, autophagy, and resistance to oxidative stress [34, 35]. Impaired glucose uptake in the diabetic heart is often linked with reduced expression or activity of the downstream intermediates in the insulin signaling pathway. In this study, the levels of p-AKT and p-Foxo1 were markedly downregulated in DCM. Decreased cardiac basal and insulin-stimulated phosphorylation of AKT and Foxo1 is evident in diabetic mouse models [36]. In our previous studies, prolonged HFD feeding of mouse models impaired AKT activation and Foxo1 phosphorylation, which resulted in persistent Foxo1 nuclear localization and activation [3, 4], consequently leading to cardiac dysfunction. Furthermore, our recent study showed a reduction in the expression of p-AKT and p-Foxo1 and in cardiac function in db/db mice [5]. KATP plays a key protective role in the heart through various signaling pathways. Specifically, genetic manipulation of cardiomyocyte insulin signaling intermediates has demonstrated that partial cardiac function rescue was achieved by upregulation of the insulin signaling pathway in diabetic hearts [37]. Similarly, a previous study has reported that the cardioprotective effect of KATP occurs at least partially by regulating the AKT-Foxo1 signaling pathway, which in turn influences the expression of PGC-1α and its downstream target genes [38]. Our recent study also showed that opening of mitoKATP increased the phosphorylation of AKT and Foxo1, but the effects of this opening were blocked by the specific AKT inhibitor, MK-2206 [5]. In our current study, Kir6.1 knockout further suppressed the phosphorylation of AKT and Foxo1 in DCM mice and increased cardiac dysfunction. On the contrary, Kir6.1 overexpression upregulated the phosphorylation of AKT and Foxo1 in DCM models and improved cardiac dysfunction both in vivo and in vitro. The above data indicate that Kir6.1 overexpression attenuates cardiac dysfunction in DCM, probably through the AKT-Foxo1 signaling pathway.
Foxo1 and its downstream targets play a key role in mitochondrial biogenesis [39]. Transient insulin stimulation activates the PI3K-AKT signaling pathway and suppresses Foxo1 activation. Inactivation of AKT through an AKT-specific inhibitor activated Foxo1. Activation of Foxo1 results in heme deficiency, limiting mitochondrial cofactor biosynthesis and ATP production [3, 4, 40]. The stability of ΔΨm is important for energy conversion. A decrease in the ΔΨm affects energy conversion, leading to cell dysfunction [41]. In our study, the AKT-specific inhibitor, MK-2206, prevented endogenous AKT activation, resulting in Foxo1 activation, decreased Kir6.1 expression and reduced ΔΨm. However, Foxo1 inactivation upregulated Kir6.1 expression and increased ΔΨm. Foxo1 promotes loss of mitochondria by activating the gene expression of hemeoxygenase-1, an enzyme that catalyzes heme degradation. Heme is an essential component of mitochondrial complexes III and IV [3, 40]. Chromatin immunoprecipitation assay demonstrated that the Kir6.1 promoter region contains a functional Foxo1-binding site. Foxo1 interacts with the promoter region of Kir6.1 for transcriptional activation. Our results indicate that the interaction between the AKT-Foxo1 signaling pathway and Kir6.1 may play a key role in the pathogenesis of DCM.