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AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

ABSTRACT: Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.

SUBMITTER: Kolwicz SC

PROVIDER: S-EPMC4817808 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

Kolwicz Stephen C SC Odom Guy L GL Nowakowski Sarah G SG Moussavi-Harami Farid F Chen Xiaolan X Reinecke Hans H Hauschka Stephen D SD Murry Charles E CE Mahairas Gregory G GG Regnier Michael M

Molecular therapy : the journal of the American Society of Gene Therapy 20150921 2

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For t ...[more]

PMID: 26388461

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Project description: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".

Dataset Information

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

Publications

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

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