Loss of the peripheral molecular clock blunts the cardiac response to mineralocorticoid induced cardiovascular disease

E.K. Fletcher,^1,2 L.M.D. Delbridge² and M.J. Young,¹ ¹Cardiovascular Endocrinology Laboratory, Prince Henry's Institute of Medical Research, Clayton, VIC 3800, Australia and ²Cardiac Phenomics Laboratory, Department of Physiology, University of Melbourne, VIC 3010, Australia.

Background: Clinical and experimental studies demonstrate that activation of the mineralocorticoid receptor (MR) in the heart leads to increased inflammation, fibrosis and endothelial dysfunction. These effects can be abrogated by MR antagonists. MR antagonists, however, have a limited use due to their side effects. Consequently the identification of cell-specific MR signaling mechanisms may allow for the development of more specific cardiac MR antagonists. One potential mechanism downstream of MR activation that may promote cardiac fibrosis is the peripheral molecular clock. The molecular clock is a transcriptional translational feed-back loop comprising of core “clock genes” which bind to and regulate numerous downstream cardiac genes. Dysregulation of the peripheral molecular clock genes in the heart leads to altered transcription of cardiac target genes, contributing to many aspects of cardiovascular disease.

Hypothesis: Activation of cardiomyocyte MR leads to dysregulation of the peripheral molecular clock resulting in cardiac inflammation, fibrosis and dysfunction.

Aim: To (i) determine whether MR-mediated cardiac fibrosis and hypertensions is dependent upon dysregulation of the peripheral molecular clock, and (ii) characterize MR dysregulation of the peripheral molecular clock in vivo.

Methods: Eight week old male wild type (WT) and ClockΔ19^mel+(CLK) mutant mice were uninephrectomised under anesthesia (Xylazil/Ketamine 10/80 mg/kg i.p. followed by analgesia Carprofen/Bupeneforin 5mg/20μg /kg i.p.) and maintained on 0.9% saline without (VEH) or with a deoxycorticosterone (DOC) 7mg/week pellet (n = 8-11) for eight weeks. Systolic blood pressure (SBP) was recorded at both four and eight weeks into the study. Heart tissue was analyzed for inflammation: macrophage and CD3+ T cell recruitment, and fibrosis: picrosirius red staining and immunohistochemistry. Quantitative RT PCR was used to assess expression of peripheral molecular clock genes and cardiac markers of fibrosis and inflammation in whole heart.

Results: Systolic blood pressure, inflammation and fibrosis were modestly elevated in VEH treated CLK mice (5% increase in SBP, 34% increase in macrophages, 13% increase in T cell numbers, and 35% increase in tissue collagen) compared to WT VEH. However, the response to DOC for each of these parameters was blunted in CLK mice compared to WT mice. PCR analysis of the core peripheral molecular clock genes revealed a significant decrease in CLOCK expression in response to DOC/salt and a significant increase in period 2 (PER2) expression. Gene expression for non-core clock members, D-albumin binding protein (DBP), and RORα in addition to Collagen III and connective tissue growth factor (CTGF) also showed increased values in VEH treated CLK mice compared to WT, but a blunted response to DOC/salt in the CLK mice.

Conclusion: These data show that although loss of a functional peripheral molecular clock alone modestly increases SBP, inflammation and fibrosis, there is a blunted cardiac response to DOC/salt mediated cardiac fibrosis and inflammation. These data suggest a role, at least in part, for MR regulation of the peripheral molecular clock in cardiovascular disease.