Brown adipose tissue (BAT), for a long time only known for its thermogenic capacity, was lately suggested to act as a modulator of cardiovascular diseases by regulating e.g. lipid clearance and glucose homeostasis. Further, BAT acts as an organ with a unique endocrine profile by secreting systemically active batokines. After cardiac ischemia/ reperfusion injury (I/R), massive sympathetic activation and catecholamine release directly impact on BAT function. Thus, a crucial role of BAT in I/R is assumed. 10-12-week-old male C57BL/6J mice (Janvier) underwent either surgical removal of BAT or sham surgery (control), three days prior I/R. Heart function was measured up to three weeks post I/R by echocardiography and the scar size was determined by Gomori-staining. Cardiac and BAT glucose metabolism was analyzed using 2HNMR.The inflammatory response, heart tissue and circulating immune cells were analyzed by flow cytometry three days post I/R. Additionally, the same experimental setup was used to perform single cell RNA sequencing of the heart, to characterize cardiac myeloid cells. Furthermore, bulk RNA sequencing was performed on BAT tissue 6 hours after either I/R injury or sham operation as control. The same setup was used to analyze the endocrine role of BAT and the supernatant of BAT was analyzed using multiplex bead assays. Finally, using a specific β3-receptor agonist, infarct size, heart function and cardiac inflammation was measured with 1HNMR.
Using the model of surgical BAT ablation, absence of BAT was shown to impair cardiac hemodynamic function over a 21-day follow up period post-I/R and to increase left ventricular scar sizes. Both, circulating as well as cardiac immune cells were increased in BAT deficient mice three days post I/R. Single cell RNA sequencing analysis revealed a proinflammatory phenotype of cardiac myeloid cells which was paralleled by an increase of cellular glycolysis. In line, 2HNMR measurement revealed transiently increased glucose fluxes in heart and BAT after I/R. The I/R itself resulted in an activation of BAT, leading to a markedly reduced secretion of proinflammatory cytokines. Moreover, the positive character of BAT after stimulation has already been evident on transcript level. Ultimately, activation of BAT by a β3-receptor agonist improved the outcome post I/R.
55Summarized, we demonstrate here that the absence of BAT significantly worsens the outcome post I/R, due to quantitatively and qualitatively altered immune cells. Cardiac myeloid cells alter their metabolism toward increased glycolysis, thereby developing a pro-inflammatory phenotype. In addition, I/R-associated activation of BAT markedly reduces the secretion of proinflammatory cytokines. Finally, by applying the beta-3-receptor agonist Mirabegron prior to I/R, a significant improvement of cardiac function after the event was recorded.