In human AC16 cardiomyocytes, we provided evidence that: i) short term (48h) high glucose treatment induced a significant increase in miRNA-21 expression and function, which was associated with increased hydrogen ion flux and energy potential dissipation without any change in energy production or increase in the expression of cellular damage genes; ii) the inhibition of miRNA-21 function induced the activation of inflammation, apoptosis pathways and compromises mitochondrial function as demonstrated by the incapacity to answer energy demand; iii) long term (7days) myocardial cells exposition to high glucose treatment evoked a decrease in miRNA-21 levels combined to an increased expression of cellular damage-related genes. In addition, a parallel depletion of the cellular energy function with secondary impairment of physiological mitochondrial function was also observed.
These results demonstrate that in human cardiomyocytes, the cardioprotective effect of miRNA-21 towards high-glucose treatment related damage is dose and time-dependent and indicate, for the first time, a potential role of miRNA-21 in molecular mechanisms underlying the myocardial metabolic flexibility, i.e., the heart's ability to adjust its substrate preference to a short-term external metabolic insult to sustain an adequate ATP production for correct cardiac contractile function.
The miRNA-21 plays a key role in many biological processes, and it is expressed in cardiomyocytes, fibroblasts, and endothelial cells, where it regulates apoptosis, inflammation, fibrosis, and metabolism. However, its role in cardiovascular diseases is still controversial (13, 16). Several previous data showed miRNA-21 to display in cardiomyocytes a protective effect toward oxidative stress via activation of PDCD4 and AP-1, two members involved in the apoptosis cascade pathway (5), and attenuates the injuries induced by palmitate (17). Indeed, miRNA-21 knockout mice displayed fibrosis and cardiac hypertrophy in response to various cardiac stresses (18). In contrast, other studies demonstrated that a high glucose treatment increased the miRNA-21 expression in cardiac fibroblast, inducing fibrosis, hypertrophy, and cardiac dysfunction (17, 19) and that elevated myocardial and bloodstream miRNA-21 levels significantly correlates with the severity of left ventricular fibrosis in aortic stenosis patients.
Interestingly, miRNA-21 has been recently demonstrated to play a critical role in the "diseased heart" by regulating myocardial energy metabolism (20). In normal conditions, to respond to the continuous demand of ATP, the heart can metabolize different substrates through mitochondrial oxidative phosphorylation, like as glucose, fatty acids, amino acids, and lactate. The main regulatory mechanisms include the uptake of substrates into cardiomyocytes through facilitated diffusion mediated by membrane-associated proteins (CD36 for fatty acids, GLUT1, and GLUT4 for glucose) and mitochondrial oxidation (21, 22). Even though the heart uses fatty acids as the primary substrate in normal conditions, a specific and delicate regulated balance exists between substrates. Increasing evidence suggests that the heart works better when it uses substrates mixture, especially fatty acids and glucose (23, 24). However, when the balance of substrates is a slant towards the predominant utilization of fatty acids or glucose, it is linked to impaired cardiac contractile function. In addition, the preferred use of one substrate regarding another increases the risk that the heart suffers from fuel toxicity, i.e., lipo- or glucotoxicity, conditions that elicit significant impairments of cardiac functioning (22, 25, 26). Specifically, glucose 6-phosphate due to a mismatch with glucose oxidation during excess glucose availability, leading to mTOR activation and increased protein synthesis resulting in cardiac hypertrophy (22, 27). In addition, long-term glucose supply and utilization are linked to a fetal transcriptional program in adult cardiomyocytes. This fetal switch is maladaptive, whereby contractile force decreases and induces ROS formation and increased glycation (28).
"Metabolic flexibility" is the capacity of the heart to adapt its substrate preference to short-term detrimental stimuli to maintain an adequate ATP production for optimal cardiac contractile function. However, persistent metabolic insult occurs when the balance of metabolic substrates is shifted towards the utilization of one substrate (either glucose or fatty acids), impairing metabolic flexibility and leading to suboptimal ATP production and impairing contractile function. Indeed, as ATP production is reduced, heart failure progresses overall (22).
However, the mechanisms underlying such metabolic flexibility and what happens during long-term increases in glucose supply and utilization are not yet fully understood.
Interestingly, in our study, short-term (2 days) exposure of cardiomyocytes to high glucose treatment elicited an up-regulation of miRNA-21, which in turn results in a protective action toward oxidative stress, inflammation, fibrosis, apoptosis. Indeed, cardiomyocytes responded to short-term high glucose concentration with a decrease of GLUT4 and PPAR-α, and increased PPAR-γ expression for downgrading glucose uptake and rising its metabolism to prevent high glucose insult. Furthermore, after a short-time human cardiomyocyte exposition to high glucose, more O2 consumption and H+ dissipation was not translated in ATP production. More intriguing, when cardiac injury persisted for a long time (7 days), the miRNA-21 levels decreased, cardiomyocytes could not protect themselves, and functional failure occurred as demonstrated by oxidative stress, inflammation, apoptosis, and fibrotic markers. Moreover, when miRNA-21 is blocked with its LNA-antagomi-RNA, basal respiration, maximal respiration, and ATP production processes are impaired in AC16 cells in a time-dependent manner.
These results suggest that cardiomyocytes might activate a compensative way to respond to the presence of abundant glucose substrate, probably through up-regulation of miRNA-21. In parallel, miRNA-21 blockage alone impedes the correct metabolic flux inside cardiomyocytes. In agreement, a recent study provides direct evidence that modulation of miR-21 levels can impact substrate utilization and mitochondrial respiration in H9C2 cells. The different substrate availability can further modulate this effect (29). Furthermore, in our study, miRNA-21 inhibition activated inflammation and apoptosis pathways, compromised mitochondrial function (as demonstrated by the reduction in O2 consumption and ATP production), thus inducing a functional failure with the inability to meet energy demand. These findings strengthen the idea that miRNA-21 presence is necessary for physiological conditions especially when the metabolic burden shifted for availability/accumulation of a predominant substrate (here glucose).
Noteworthy, long-term exposure to high glucose treatment, a condition associated with downregulation of miRNA-21, definitively compromises the cellular metabolism as demonstrated by the reduction of spare capacity, indicating that cardiomyocytes lost flexibility and cell fitness (30). These data are also supported by the decrease in O2 consumption, ATP production, and the increase of H+ dissipation with consequent activation of antioxidant defense. Moreover, the up-regulation of PPAR-α and the down-regulation of PPAR-γ suggest that cardiomyocytes try to revert the metabolic imbalance in favor of fatty acid oxidation. Still, this effort helps to increase ROS generation to toxic levels, leading to subsequent activation of the antioxidant defense system (31).
These results highlight the complexity of factors that regulate metabolic pathways in the cardiac cells and suggest that, under hyperglycemic conditions, the cardiomyocytes, before irreversible damage, realize an early and late adaptation that recalls the two preliminary phases of diabetic cardiomyopathy. Indeed, in an early phase, the cell recognizes a protective mechanism against the high exposure of high glucose-mediated by miRNA-21; subsequently, as a consequence of miRNA-21 downregulation, the cell cannot counteract the damage and goes into functional metabolic deficiency. Moreover, even when glucose is not the primary energy source, the inhibition of miRNA-21 function alone shows time-dependent effects. Indeed, at 24h from the LNA treatment, cardiomyocytes maintain the metabolic flow while a decrease in the total cellular ATP still uncompensated by the non-mitochondrial oxygen consumption pathways over a longer time incubation was observed. As a whole, our data underline the importance of assessing alteration in mitochondrial respiration in addition to genes expression and points out the crucial role of miR-21 as a new potential target for metabolic intervention aimed at securing sufficient ATP production to sustain organ function both in response to short- and long-term metabolic insult.