Age- and Sex-Dependent Caveolar Gene Expression in Normoxic and Post-Ischemic Mouse Hearts

Abstract

Age and sex modify myocardial I-R tolerance with caveolar invaginations in the plasma membrane shown to be important determinants of ischemic tolerance as well as responsiveness to cardioprotective stimuli. Caveolar membrane domains are formed by variety of coat proteins including those of caveolins, cavins and Popeye domain-containing family of proteins. Expression of these proteins are unclear in the aging male and female myocardium. Expression of caveolae-associated transcripts and proteins were assayed in normoxic and post-ischemic (20 min ischemia/60 min reperfusion) myocardium from 8-, 16-, 32- and 48-week old male and female mice (C57Bl/6). Using cultured HL-1 cells, the reliance of acute A1 Adenosine Receptor agonism on caveolae was also assessed, a known stress tolerance determinant that reside in caveolae. Significant down-regulation of chief caveolae transcripts Cav3 and Cavin1 correlated with 38%-52% reductions in Caveolin-3 & Cavin-1 protein in normoxic middle-aged hearts (respectively), congruent with age-related repression of caveolins in other tissue types. For females, caveolae-related genes were largely stable with aging which were consistent with previous findings that middle-aged female hearts display increase stress tolerance as measured by LDH release following I-R. Age thus appears to suppress transcription/expression of caveolar proteins critical to myocardial responses to stress and protective stimuli, an effect more pronounced in male vs. female myocardium. These age-dependent shifts in caveolar coat protein expression was associated with exaggerated contractile dysfunction associated evident at 32- and 48-weeks (vs. 8 weeks) in male hearts, but not associated with impaired recovery in 32 and 48-weeks (vs. 8 and 16 weeks) in females. In HL-1 cardiomyocytes subject to simulated ischemia-reperfusion, acute A1AR agonism with 100 nM 2-chloro-N6-cyclopentyadenosine (CCPA) resulted in cytoprotection evident by significantly decreased LDH release (by 20%), which was abolished by caveolae & cholesterol depletion agent methyl-{beta}-cyclodextrin (M{beta}CD, 1 mM), suggesting caveolar involvement in protective signaling. HighlightsO_LIAged-related reduction in ischemic tolerance evident in middle-aged male and female heart before "aged" phenotype typically used in aging studies. C_LIO_LIDeclining ischemic tolerance coincident with age-related shifts in Cav1, Cav3, Cavin1 and Popdc1 mRNAs, and reduced Caveolin-3 and Cavin-1 protein in males. C_LIO_LIIn the aging female myocardium crucial caveolar coat transcripts Cav3, Cavin1 and Popdc1 were largely stable with aging and I-R, consistent with reports of enhanced stress tolerance in the female heart & role of caveolae in promoting stress resistance. C_LIO_LITop predicted microRNA regulators of Cav3 transcript microRNA-22 and microRNA-101b did not inversely correlate with observed Cav3 mRNA expression profiles in the aging heart. C_LIO_LIIn simulated ischemia-reperfused cultured cardiomyocytes, acute A1AR agonism markedly decreased cell injury, this protection was abolish by cholesterol & caveolae depleting agent M{beta}CD which also enhanced cell death, highlighting the role of caveolae in endogenous ischemic tolerance. C_LI