The Cardiac Circadian Proteome and Transcriptome: a Novel Paradigm Characterizing Cardiovascular Health and Disease
Circadian rhythms in cardiovascular physiological and molecular processes are essential for cardiac health in the diurnal (day/night) environment. The circadian clock mechanism found in most peripheral tissues, including the heart, controls circadian rhythms by regulating ~24h cycles in gene expression. Previous studies have shown that 9-13% of cardiac genes exhibit circadian rhythms in mRNA levels. However, these studies are insufficient, because proteins, and not mRNA, carry out most biological processes in the cell. This thesis investigated diurnal cardiac proteome in health and disease and the mechanisms underlying protein abundance rhythms. The first study introduced a cardiovascular circadian proteomics field of investigation and described the application of two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry for detecting temporal changes in protein abundance in healthy and diseased mouse hearts. The second study quantified the healthy murine cardiac proteome across a 12:12 light/dark (LD) cycle by 2D-DIGE and revealed that 90 out of 1147 (~8%) spots exhibited significant rhythmic changes in abundance. Half of the identified diurnal proteins had statistically significant changes in expression of their corresponding mRNA. Alterations in cardiac proteome were detected in cardiomyocyte specific clock mutant (CCM) mice, consistent with the regulation by the clock mechanism. Diurnal disruption (10:10 LD) altered rhythmic protein expression, and cardiac contractility on the Langendorff apparatus. Diurnal proteome could also be important in heart disease, as 70 out of 998 (7%) of detected cardiac proteins and peripheral blood cytokines exhibited diurnal changes in abundance in a murine model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Diurnal rhythms in myofilament ATP consumption were reduced in TAC hearts compared to controls. The third study developed a bioinformatics framework to investigate clock-controlled transcription in the heart. Putative clock-controlled genes exhibited a characteristic circadian expression pattern and had conserved E-box cis enhancer elements in their promoters. The bioinformatics approach was experimentally validated by demonstrating that the circadian clock regulated rhythmic expression of the titin cap gene. Collectively, this thesis supports a novel paradigm in cardiovascular biology: temporal gene and protein expression is an important factor affecting cardiovascular health and disease.