The present study shows that genetic loss of insulin signaling leads to accelerated LV dysfunction following myocardial infarction. LV dysfunction is associated with reduced rates of mitochondrial oxygen consumption in response to the FA substrate palmitoyl carnitine. Although there is a general repression of FAO genes in the post-MI LV in control mice, loss of insulin signaling is associated with reduced expression of many FAO and OXPHOS genes at baseline. However, there is progressive reduction in levels of the transcriptional regulator of FAO, PPAR-α and additional declines in expression levels of MCAD, LCAD, CPT2 and ETFDH. Thus loss of insulin signaling in the heart may limit FA utilization during remodeling of the LV following myocardial infarction. In addition, there is further repression of GLUT4 expression in the LV of CIRKO mice post-MI. GLUT4 is an important mediator of basal myocardial glucose uptake in vivo [12
], thus reduced GLUT4 expression might further limit substrate availability thereby contributing to impaired LV function following MI. Although we focused primarily on the potential role of mitochondrial dysfunction in impairing LV function in CIRKO hearts following MI, our studies revealed additional pathogenic mechanisms. Invasive LV catheterization revealed reduced −dP/dt and papillary muscle preparations revealed impaired relaxation kinetics. Although diastolic relaxation is an energy requiring process, which could be impaired in the context of reduced mitochondrial energetics, defects in calcium re-uptake or myosin isoform switching could also contribute to impaired diastolic relaxation, as was confirmed by increased expression of the slow-twitch β-myosin isoform and reduced expression of SERCA2 in CIRKO hearts following MI.
Many human studies confirm that outcomes of ischemia/reperfusion and adaptations of diabetic hearts to ischemia are impaired [1
]. Ischemic heart disease accounts for more than 50% of deaths in diabetic patients and mortality from acute myocardial infarction is increased 2-fold in diabetic patients compared with non-diabetic individuals [26
]. Metabolic disturbances associated with diabetes mellitus such as hyperglycemia, hyperlipidemia and insulin resistance, have been postulated to contribute to the incidence and outcomes of myocardial ischemia in diabetes [6
], however the specific contribution of insulin resistance or loss of insulin action per se
have been difficult to elucidate. Studies in animal models of genetic or diet-induced insulin resistance and obesity, have illustrated adverse outcomes following in vivo coronary artery ligation (reviewed in [30
]). And although in some of these models, myocardial insulin resistance has been reported, it remains uncertain the extent to which impaired myocardial insulin action may directly contribute to adverse outcomes following myocardial infarction. The present study utilized a model with a genetic deficiency in insulin signaling that was isolated to cardiomyocytes and suggests that impaired myocardial insulin signaling might independently contribute to adverse LV remodeling following myocardial infarction. Importantly, vascular smooth muscle and endothelial insulin signaling are not affected in this model
The mechanisms by which impaired insulin signaling leads to mitochondrial dysfunction are complex. We have recently reported that in CIRKO hearts, mitochondrial dysfunction in non-stressed hearts is due in part to oxidative stress and a global reduction in mitochondrial content of FAO proteins and critical subunits of the pyruvate dehydrogenase complex, that are due to reduced expression of FAO genes and their regulator PPAR-α and the PDH E1α1 subunit [31
]. Moreover, PI3K signaling may play an important role in increasing mitochondrial FAO capacity in the context of physiological cardiac hypertrophy [32
]. Thus impaired insulin-PI3K signaling reduces mitochondrial metabolic capacity that likely renders them more susceptible to progressive mitochondrial dysfunction during post-MI LV remodeling. Although our data raises the possibility of a causal relationship between mitochondrial dysfunction and accelerated LV dysfunction in the remodeled LV of CIRKO mice following myocardial infarction, an alternative possibility is that mitochondria in CIRKO mice are sensitized to the increase in oxidative stress and tissue hypoxia that characterizes the remodeling LV. Indeed, we observed a striking increase in expression of HIF-1α mRNA in CIRKO hearts post MI. Oxidative stress has been shown to increase the binding of NFK
B to the HIF-1α promoter thereby increasing its expression [33
]. Moreover, HIF-1α has been shown to directly repress the expression of PPAR-α in epithelial cells [36
]. Thus it is possible that an initial impairment in mitochondrial dysfunction in CIRKO hearts could be amplified during the process of LV remodeling, which sets up a vicious cycle leading to an acceleration of mitochondrial and contractile dysfunction following MI in CIRKO hearts.
Some recent studies have examined the mitochondrial adaptations to LV hypertrophy following pressure overload and to LV remodeling following myocardial infarction. In general, compensated pressure overload hypertrophy is associated with reduced expression of PGC-1α and its co-activated genes, leading to reduced FAO and OXPHOS capacity [37
]. In addition, many studies have reported repression of PPAR-α levels in the failing heart [41
]. In contrast, in heart failure following coronary artery ligation, mitochondrial function has been reported to be preserved and associated with normal PPAR-α expression [45
]. These differences might reflect differences in model systems and duration or severity of heart failure. Our data in WT-MI hearts, in which mitochondrial function was normal, is consistent with these latter reports. It is important to note that in the present study, heart failure had not yet developed in WT mice but there was compensated cardiac hypertrophy, with reduction in expression of PGC-1α, OXPHOS and FAO genes. In contrast, CIRKO-MI mice had significant LV dysfunction and although they shared many of the transcriptional changes observed in WT-MI mice, there was clear evidence of mitochondrial dysfunction. Our transcriptional analyses suggest that this could be the result of a specific reduction in PPAR-α expression, which might have contributed to the additional reduction in expression of the beta oxidation enzymes LCAD and MCAD, CPT2, which would limit mitochondrial FA-acyl CoA uptake, and ETFDH, which might limit the delivery of reducing equivalents generated by beta oxidation to the electron transport chain. These mechanisms could all contribute to the observed defect in palmitoyl carnitine supported mitochondrial oxygen consumption. Given that FA oxidation generates up to 70% of myocardial ATP, the reduction in mitochondrial FA oxidative capacity could plausibly contribute to impaired LV function following MI in CIRKO hearts.
Uncoupling protein (UCP2) expression was increased in hearts of WT-MI, despite repression of PGC-1α. In CIRKO hearts, UCP2 expression was induced in non-stressed hearts and remained increased post MI, despite progressive repression of PPAR-α. Cardiac UCP2 expression is regulated by PPAR-α dependent and independent mechanisms [47
]. Thus PPAR-α independent pathways primarily regulate UCP2 expression following ischemia in these models. It is generally accepted that the ischemic myocardium generates reactive oxygen species (ROS) from mitochondrial sources in response to ischemic injury, (reviewed in [48
]). Activation of uncoupling proteins has been suggested to play a cardioprotective during ischemia and reperfusion [51
]. UCP2 expression and activity may be regulated by ROS and a putative mechanism by which this may occur in the heart in the context of ischemia may involve activation of ASK1/JNK-p38/CREB-NFκB signaling pathways [49
]. We have recently reported that oxidative stress is increased in non-stressed CIRKO hearts [31
]. Activation of uncoupling proteins could contribute to reduced mitochondrial energetics [55
]. Future studies will be required to determine if mitochondrial uncoupling could also contribute to the reduction in mitochondrial energetics in insulin resistant hearts following myocardial ischemia.
Our study has some limitations. CIRKO mice exhibited increased acute mortality following coronary artery ligation. We do not know the mechanisms for this interesting phenomenon but speculate that the initial early mortality in the immediate peri-MI period could be due to arrhythmias. CIRKO mice have previously been shown to exhibit attenuated cardiac potassium currents, a feature which could promote arrhythmias [56
]. Future studies will probe these mechanisms further. Because of challenges in generating large numbers of CIRKO mice, we did not measure substrate metabolism in isolated hearts or activity levels of beta-oxidation enzymes or of carnitine palmitoyl transferase, or activity of intracellular survival or stress-activated kinases. It is possible that absence of additional insulin signaling-mediated pro-survival mechanisms [57
] could have contributed to the accelerated rate of LV dysfunction or excess mortality. Genetic deletion of insulin signaling is perhaps more severe than the more partial degrees of cardiac insulin resistance that might occur in individuals with diabetes, obesity or insulin resistance. Thus additional studies in models with selective, but partial impairment in myocardial insulin action will be required to determine the consequences of lesser degrees of insulin resistance on the myocardial adaptations following myocardial infarction.
In summary, the present study demonstrates that impaired insulin signaling in cardiomyocytes accelerates adverse post-MI LV dysfunction. These changes are associated with a rapid decline in mitochondrial function that parallels a specific reduction in the expression of the transcriptional regulator PPAR-α and additional reduction in expression of genes whose products determine mitochondrial beta-oxidation capacity. Thus, insulin signaling might play an important role in sustaining LV metabolic capacity in post-MI LV remodeling.