Myocardial stunning is the transient cardiac dysfunction that follows brief episodes of ischemia and reperfusion without associated myocardial necrosis. Currently, there is limited knowledge about its cellular and biochemical mechanisms. In order to better understand the underlying mechanisms of contractile dysfunction associated with the stunning, comprehensive proteomic studies using 2-D DIGE were performed using a regional stunning model in canine heart. Cardiac myosin binding protein C (cMyBP-C), a regulatory myofilament protein associated with the thick filament, and nebulette, a thin filament associated protein, were differentially expressed. Phosphoprotein specific staining indicated both protein changes were due to phosphorylation. Subsequent phosphorylation mapping of canine cMyBP-C using IMAC and MS/MS identified five phosphorylation sites, including three novel sites. In order to further evaluate this finding in a different model, cMyBP-C phosphorylation was examined in a rat model of global stunning. In the rat model, stunning was associated with increased phosphorylation of cMyBP-C at a critical calcium/calmodulin-dependent kinase II site, and the increased phosphorylation was largely inhibited when stunning was prevented by either ischemic preconditioning or reperfusion in the presence of low-calcium buffer. These data indicate cMyBP-C phosphorylation plays an important role in myocardial stunning.

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The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a ␤-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca 2؉ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased ␤ myosin heavy chain expression compared to age matched nontransgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.Troponin I (TnI), 2 the inhibitory domain of the troponin (Tn) complex, is an essential protein involved in regulation of the actin-myosin interaction responsible for striated muscle contraction. Troponin I is encoded by three muscle genes expressing fast skeletal, slow skeletal, and cardiac TnI (cTnI) isoforms (1), with the cTnI gene the most recently evolved (2). Unlike the fast and slow skeletal isoforms, the cTnI isoform has evolved to contain a unique 32 amino acid N-terminal extension with the function of modulating cardiac contraction. In response to ␤-adrenergic-induced stimulatory G-protein (G s ␣) activation of protein kinase A (PKA), Ser-23/24 of the cTnI N-terminal extension are bis-phosphorylated (3). Phosphorylation of these residues alters cTnI structure diminishing the cTnI N-terminal interaction with troponin C (TnC) to decrease Ca 2ϩ -sensitive activation of muscle contraction (4, 5) and increase myosin cross-bridge kinetics (6 -8). Functionally, this structural change in the modulatory cTnI N-terminal extension is essential to increase the rate of myocardial relaxation and maintain adequate diastolic filling during the increased heart rate of ␤-adrenergic stimulation to meet increased cardiac demand. Non-physiological truncations removing the cTnI N-terminal extension result in a cTnI molecule that exhibits similar structural and functional al...