The long-term goal of this work is to implement novel gene-based therapeutics to reverse contractile performance deficits in the failing human heart in vivo. This proposal is focused on troponin I (TnI), the inhibitory subunit of the troponin complex and molecular switch to cardiac contraction. The focus of this proposal on advances in troponin function in the diseased heart has considerable health relevance and, accordingly, well serves the mission of the NIH. Novel preliminary evidence is presented on troponin-modified myofilaments dictating myocyte Ca2+ handling through a direct effect on sarcoplasmic reticulum (SR) Ca2+ load. The overarching hypothesis that guides this proposal is myofilament function, by virtue of unique properties of histidine modified TnI at codon 164, uniquely and directly "tunes" SR Ca2+ content independent of effects on canonical regulators of SR Ca2+ load, including SERCA2a -dependent rapid Ca2+ sequestration. We further posit that novel bio-engineered adeno-associated viral vectors, together with our new high fidelity hypoxia responsive gene switch system, will permit translation of this proposal's molecular/cellular advances (Aim1) directly to the beating heart in vivo in pre-clinical studies in living mammals (Aim 2).
The Specific Aims are:
Aim 1. To determine the molecular mechanism underlying optimization of global cardiac myocyte Ca2+ handling between the myofilaments and sarcoplasmic reticulum in vitro and in vivo by single histidine-modified troponin. Hypothesis: Real time sensing of the patho-physiological biochemical milieu of the healthy and diseased myocyte by the troponin modified sarcomeres will have a dominant effect to directly decrease the Ca2+ load requirements of the SR, including conditions of accelerated SR Ca2+ influx by an increased SERCA2a/PLN ratio.
Aim 2. To implement a novel evolutionary-directed bio-engineered AAV vector and double oxygen genetic sensing system for optimization of efficient systemic gene delivery of modified troponins in the diseased heart in vivo. Hypothesis: Compared to wild-type striated muscle tropic rAAV serotypes AAV1, pseudo-typed AAV6, AAV8 and AAV9, the novel bio-engineered rAAV capsid M41 with O2 sensing genetic elements will lead to higher cardiac tropism and greater efficiency resulting in hypoxia/ischemia sensitive long-term constitutive expression of modified TnIs in the cardiac sarcomeres of small mammals.
Ischemic cardiomyopathy is a major problem in the United States. This proposal focuses on a modified heart muscle protein troponin to enhance heart pump performance in the ischemic heart. Therefore the health relevance of the proposal is substantial and highly significant to the mission of the National Institutes of Health.
|Thompson, Brian R; Metzger, Joseph M (2014) Cell biology of sarcomeric protein engineering: disease modeling and therapeutic potential. Anat Rec (Hoboken) 297:1663-9|
|Thompson, Brian R; Houang, Evelyne M; Sham, Yuk Y et al. (2014) Molecular determinants of cardiac myocyte performance as conferred by isoform-specific TnI residues. Biophys J 106:2105-14|
|Wang, Wang; Asp, Michelle L; Guerrero-Serna, Guadalupe et al. (2014) Differential effects of S100 proteins A2 and A6 on cardiac Ca(2+) cycling and contractile performance. J Mol Cell Cardiol 72:117-25|
|Martindale, Joshua J; Metzger, Joseph M (2014) Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization. J Mol Cell Cardiol 67:26-37|
|Wang, Wang; Barnabei, Matthew S; Asp, Michelle L et al. (2013) Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. Nat Med 19:305-12|
|Palpant, Nathan J; Bedada, Fikru B; Peacock, Brandon et al. (2011) Cardiac disease in mucopolysaccharidosis type I attributed to catecholaminergic and hemodynamic deficiencies. Am J Physiol Heart Circ Physiol 300:H356-65|
|Palpant, Nathan J; Metzger, Joseph M (2010) Aesthetic cardiology: adipose-derived stem cells for myocardial repair. Curr Stem Cell Res Ther 5:145-52|
|Palpant, Nathan J; Houang, Evelyne M; Delport, Wayne et al. (2010) Pathogenic peptide deviations support a model of adaptive evolution of chordate cardiac performance by troponin mutations. Physiol Genomics 42:287-99|
|Fomicheva, Ekaterina V; Turner, Immanuel I; Edwards, Terri G et al. (2008) Double oxygen-sensing vector system for robust hypoxia/ischemia-regulated gene induction in cardiac muscle in vitro and in vivo. Mol Ther 16:1594-601|
|Palpant, Nathan J; Day, Sharlene M; Herron, Todd J et al. (2008) Single histidine-substituted cardiac troponin I confers protection from age-related systolic and diastolic dysfunction. Cardiovasc Res 80:209-18|
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