title: Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation.
abstract: We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca(2+)-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca(2+) fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca(2+)], indicating increased contractility is primarily due to increased myofilament activity without altering Ca(2+) release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca(2+) activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin-myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca(2+). This article is part of a Special issue entitled "Possible Editorial".
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: F S
authors.initials: FS
authors.lastname: Korte
authors.forename: Jin
authors.initials: J
authors.lastname: Dai
authors.forename: Kate
authors.initials: K
authors.lastname: Buckley
authors.forename: Erik R
authors.initials: ER
authors.lastname: Feest
authors.forename: Nancy
authors.initials: N
authors.lastname: Adamek
authors.forename: Michael A
authors.initials: MA
authors.lastname: Geeves
authors.forename: Charles E
authors.initials: CE
authors.lastname: Murry
authors.forename: Michael
authors.initials: M
authors.lastname: Regnier
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D009200:Myocardial Contraction / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics*
chemical_list: D003838:Deoxyadenine Nucleotides; D012333:RNA, Messenger; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate; D002118:Calcium