The overall goal of this proposal is to determine if energetically efficient positive inotropy can be produced by increasing the calcium sensitivity of the contractile apparatus. This will be accomplished by studying transgenic mice with mutant troponin I lacking protein kinase A phosphorylation sites, or overexpressed beta-tropomyosin. Also, to determine if energetically inefficient negative inotropy can be prevented, mutant troponin I that lacks protein kinase C phosphorylation sites will be expressed in transgenic mice. These studies may have direct implications for future therapies for congestive heart failure. Currently available positive inotropes, increase cytosolic calcium, and thereby increase oxygen consumption, resulting in energetically inefficient increases in inotropy. However, there is now a substantial body of literature suggesting that calcium sensitizing drugs, which cause positive inotropy without increasing cytosolic calcium, limit the increase in oxygen consumption. Several problems remain with these agents, including uncertainty about the site of action, and also they may have undesirable effects on the coronary and peripheral vasculature.
In Specific Aim 1 we will characterize the relationship between developed pressure, oxygen consumption and intracellular calcium in the perfused mouse heart, which will form the basis for the following studies.
In Specific Aims 2 and 3, transgenic mice which are predicted to have increased calcium sensitivity based on in-vitro studies will be studied. The first overexpresses beta-tropomyosin, and has already been generated. The second expresses mutant troponin I, which lacks the protein kinase A phosphorylation site, and we have identified several founders for this. Protein kinase C can mediate negative effects on inotropy, especially through phosphorylation of sites on troponin I, distinct from the protein kinase A phosphorylation sites.
In Specific Aim 4, a transgenic mouse expressing mutant troponin I, lacking the protein kinase C phosphorylation sites is predicted to have a greater preservation of contractile function in response to interventions that activate protein kinase C. Preliminary data from this transgenic model are presented supporting this hypothesis. In this submission, a program of didactic training, mentorship, and research development is proposed, structured to develop the candidate's research training to eventual full independent status.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL003826-01A1
Application #
2854220
Study Section
Special Emphasis Panel (ZHL1-CSR-K (F2))
Project Start
1999-07-01
Project End
2004-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
MacGowan, Guy A; Kirk, Jonathan A; Evans, Caroline et al. (2006) Pressure-calcium relationships in perfused mouse hearts. Am J Physiol Heart Circ Physiol 290:H2614-24
MacGowan, Guy A; Rager, Jennifer; Shroff, Sanjeev G et al. (2005) In vivo alpha-adrenergic responses and troponin I phosphorylation: anesthesia interactions. J Appl Physiol 98:1163-70
MacGowan, Guy A; Evans, Caroline; Hu, Tom C-C et al. (2004) Troponin I protein kinase C phosphorylation sites and ventricular function. Cardiovasc Res 63:245-55
McNamara, Dennis M; MacGowan, Guy A; London, Barry (2002) Clinical importance of beta-adrenoceptor polymorphisms in cardiovascular disease. Am J Pharmacogenomics 2:73-8
Du, C; MacGowan, G A; Farkas, D L et al. (2001) Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching. Cell Calcium 29:217-27
Du, C; MacGowan, G A; Farkas, D L et al. (2001) Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory. Biophys J 80:549-61
MacGowan, G A; Du, C; Koretsky, A P (2001) High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition. BMC Physiol 1:12
MacGowan, G A; Du, C; Cowan, D B et al. (2001) Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites. Am J Physiol Heart Circ Physiol 280:H835-43
MacGowan, G A; Panzak, G; Murali, S (2001) Exercise-related ventilatory abnormalities are more specific for functional impairment in chronic heart failure than reduction in peak exercise oxygen consumption. J Heart Lung Transplant 20:1167-73
MacGowan, G A; Du, C; Glonty, V et al. (2001) Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy. J Biomed Opt 6:23-30

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