Calcineurin and its regulator, RCAN1, confer time-of-day changes in susceptibility of the heart to ischemia/reperfusion

Abstract

Many important components of the cardiovascular system display circadian rhythmicity. In both humans and mice, cardiac damage from ischemia/reperfusion (I/R) is greatest at the transition from sleep to activity. The causes of this window of susceptibility are not fully understood. In the murine heart we have reported high amplitude circadian oscillations in expression of the cardioprotective protein regulator of calcineurin 1 (Rcan1). This study was designed to test whether Rcan1 contributes to the circadian rhythm in cardiac protection from I/R damage. Wild type (WT), Rcan1 KO, and Rcan1-Tg mice, with cardiomyocyte-specific overexpression of Rcan1, were subjected to 45 minutes of myocardial ischemia followed by 24 hours of reperfusion. Surgeries were performed either during the first two hours (AM) or the last two hours (PM) of the animal’s light phase. The area at risk was the same for all genotypes at either time point; however, in WT mice, PM-generated infarcts were 78% larger than AM-generated infarcts. Plasma cardiac troponin I levels were likewise greater in PM-operated animals. In Rcan1 KO mice there was no significant difference between the AM- and PM-operated hearts, which displayed greater indices of damage similar to that of PM-operated WT animals. Mice with cardiomyocyte-specific over expression of human RCAN1, likewise, showed no time-of-day difference, but had smaller infarcts comparable to those of AM-operated WT mice. In vitro, cardiomyocytes depleted of RCAN1 were more sensitive to simulated I/R and the calcineurin inhibitor, FK506, restored protection. FK506 also conferred protection to PM-infarcted WT animals. Importantly, transcription of core circadian clock genes was not altered in Rcan1 KO hearts. These studies identify the calcineurin/Rcan1-signaling cascade as a potential therapeutic target through which to benefit from innate circadian changes in cardiac protection without disrupting core circadian oscillations that are essential to cardiovascular, metabolic, and mental health.

Calcineurin signaling in the heart: The importance of time and place

“…Expression of the Rcan1.4 isoform is under calcineurin/NFAT control and thus functions as a feedback inhibitor of calcineurin [88]. Expression of a cardiomyocyte-specific α MHC-Rcan1 transgene protects mice from a diversity of pathological stresses including pressure overload, damage from ischemia-reperfusion (I/R) and pathological remodeling following a myocardial infarction [5, 6, 89]. The hearts of animals deficient for RCAN1 ( Rcan1 −/− ) are more susceptible to damage from I/R [89, 90].…”

“…Expression of a cardiomyocyte-specific α MHC-Rcan1 transgene protects mice from a diversity of pathological stresses including pressure overload, damage from ischemia-reperfusion (I/R) and pathological remodeling following a myocardial infarction [5, 6, 89]. The hearts of animals deficient for RCAN1 ( Rcan1 −/− ) are more susceptible to damage from I/R [89, 90]. Surprisingly, the hearts of Rcan1 −/− mice are smaller and have a blunted hypertrophic response to pressure overload [90, 91].…”

“…The peak in calcineurin activity immediately precedes a time of day when the heart is less susceptible to damage from I/R [275, 276]. These diurnal changes in susceptibility appear to be mediated by changes in RCAN1 levels, as Rcan1 −/− mice no longer display this temporal window of protection [89]. …”

Glucocorticoid-mediated co-regulation of RCAN1-1, E4BP4 and BIM in human leukemia cells susceptible to apoptosis

“…Indeed, RCAN1 expression is regulated in a circadian fashion, in the same phase as E4BP4 , according to a study that did not distinguish between RCAN1-1 and RCAN1-4 [22]. In cardiomyocytes, the oscillatory expression of RCAN1-4 , but not RCAN1-1 , serves as a cardioprotector and a vital mediator of the circadian rhythmicity of cardiovascular susceptibility to ischemia/reperfusion injury [23, 24]. …”

“…Overexpression of RCAN1 has also been shown to activate caspase-3 activity to induce apoptosis [20]. In some models, such as cardiomyocytes, calcineurin facilitates Ca 2+ dependent apoptosis, hence activation of RCAN1-4 has been shown to protect cells from apoptosis [23, 24]. …”