Effects of glucose and insulin resistance on gene expression profiling in the isolated working rat heart

We investigated the isolated working rat heart as a model to study early transcriptional remodeling induced by cardiac surgery with cardiopulmonary bypass (CPB). Because the mechanisms linking stress hyperglycemia and insulin resistance to increased peri-operative morbidity and mortality in the setting of open-heart surgery are still unclear, we also used this model to determine the transcriptional effects of exogenous glucose supply in absence or in presence of a preexisting state of myocardial insulin resistance. Male Sprague Dawley rats (200-224g) were kept on chow diet for 8 to 10 weeks, or fed a high-sucrose diet to induce myocardial insulin resistance. Following the feeding period, hearts were recovered, arrested in ice-cold Krebs-Henseleit (KH) buffer and subjected to 60 minutes normothermic reperfusion in the working mode with KH buffer supplemented with non-carbohydrate substrates plus glucose (25 mM) or mannitol (25 mM; osmotic control). Compared to non-perfused hearts, perfused hearts from chow-fed rats displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, cell proliferation, and apoptosis. The same biological pathways were activated in hearts from insulin resistant rats, with 109 genes (mannitol group) and 70 genes (glucose group) up-regulated more than twofold when compared to the non-perfused hearts from high-sucrose diet-fed rats. The changes were highly similar to gene alterations occurring in the right atrium and left ventricle of open heart surgery patients, and included the up-regulation of the three master regulators of metabolic reprogramming MYC, NR4A1 and NR4A2. Targeted pathway analysis revealed an up-regulation of metabolic processes associated with the proliferation and activation of tissue-resident macrophages and fibroblasts. Although the remodeling occurred independently from glucose, glucose further increased the expression of a subset of genes associated with polarization of tissue reparative M2 macrophages. However, glucose failed to enhance the expression of M2-related markers in the isolated heart from rats rendered insulin resistant by high-sucrose feeding. These results expose the cellular components of the heart as a significant source of proinflammatory mediators released in response to stress associated with cardiac surgery with CPB, and suggest a major role for glucose as a signal in the determination of resident cardiac macrophage polarization. Changes in myocardial gene expression induced by cold arrest and 60 min normoxic and normothermic reperfusion in presence of 25 mmol/L glucose or mannitol (osmotic control) were determined for hearts from chow-fed (insulin sensitive) or high-sucrose diet-fed rats (insulin resistant) by comparing to non-perfused rat hearts from the same feeding groups. Each dietary group of rats (chow-fed or high-sucrose diet-fed) included 3 subgroups of hearts (non-perfused, perfused with glucose, perfused with mannitol), with 5 biological replicates per subgroup – Therefore, the experiment included 30 biological samples total.