Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCε phosphorylation

Scruggs, Sarah B.; Walker, Lori A.; Lyu, Theodore; Geenen, David L.; Solaro, R. John; Buttrick, Peter M.; Goldspink, Paul H.

doi:10.1016/j.yjmcc.2005.12.009

Cited by 58 publications

(59 citation statements)

References 31 publications

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“…6. A: representative immunoblots of cTnI phosphospecies in CON (n ϭ 8), POLVH (n ϭ 8), and MICHF (n ϭ 8) ventricular samples separated by nonequilibrium isoelectric focusing as described previously (1,35). A detailed description is also provided in METHODS.…”

Section: Discussionmentioning

confidence: 99%

“…One-dimensional, nonequilibrium, isoelectric focusing (1D-IEF) analysis of cTnI-phosphorylated species was executed as described earlier (18,35). Briefly, ventricular myocardium frozen at Ϫ80°C was homogenized in buffer containing 8 M urea, 2.5 M thiourea, 4% CHAPS, 0.5% ampholytes (3-10), 2 mM EDTA, and 2 mM tributylphosphine and protease inhibitor cocktail (Sigma Aldrich).…”

Section: Methodsmentioning

confidence: 99%

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Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

Belin¹,

Sumandea²,

Kobayashi³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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(LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC50 to levels observed in failing myocytes. The Fmax parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca 2ϩ sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI. left ventricle; troponin; phosphorylation; cardiac disease CONGESTIVE HEART FAILURE (CHF) is characterized by reduced ventricular pump function, which is due, in part, to cardiac myocyte dysfunction. It has been widely reported that Ca 2ϩ homeostasis is impaired in CHF (14). However, whether depressed myofilament function contributes to reduced ventricular myocyte contractility in CHF is less clear (5). Studies probing myofilament activation in failing human myocardium must be interpreted with caution because tissue quality, pharmacological treatment, and brain death of donors may confound experimental findings (15,30,44). For these reasons, investigators have employed animal models that allow for the study of myofilament function under more carefully controlled circumstances. For instance, studies in the pacing-induced canine model of CHF indicate that the myofilaments generate more force for a given level of activator Ca 2ϩ (increased Ca 2ϩ sensitivity) compared with controls (45). Examination of myofilament activity in the spontaneously hypertensive heart failure prone (SHHF) rat demonstrates that myofilament function is either augmented or unchanged depending on when studies are performed during the disease progression (32). In contrast, Pérez and coworkers (31) found reduced myofilament function in right ventricular (RV) trabeculae of the SHHF rat. Similarly, de Tombe et al. (7) have also shown reduced myofilament function in RV trabeculae obtained from rats with large left ventricular (LV) infarcts and in skinned RV myocytes isolated from rats with chronic RV hypertrophy induced by pulmonary artery banding (9). However, the impact of experimental LV hypertrophy (LVH) or CHF on myofilament function in the more clinically relevant left ventricle has not been carefully studied. The molecular basis for altered myofilament function in LVH and CHF likely involves changes in thick and thin filament proteins. It has been reported that protein kinase C (PKC) is upregulated in cardiac disease (6,13,43). In addition, recent work from our group indicates that PKC-mediated phosphoryla...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

Belin¹,

Sumandea²,

Kobayashi³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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“…Kokubu and co-workers showed that 0.1% TFA, 50% ACN as IMAC loading buffer reduced the level of nonspecific binding and improved the specificity of the method toward phosphopeptides [56]. Yet another approach to improve the performance of IMAC is to reduce the complexity of the peptide samples by prefractionation using methods such as IEF [57][58][59][60], Ion Exchange Chromatography [41,43,46,61] or hydrophilic interaction chromatography (HILIC) [62] prior to IMAC purification.…”

Section: Immobilized Metal Ion Affinity Chromatographymentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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“…For long-term storage, cardiac tissues may be treated with TCA to abolish all enzymatic activities [20]. During the process of protein extraction, protease and phosphatase inhibitors, as well as reducing reagents, should be used, and extended exposure to elevated temperatures should be avoided.…”

Section: Sample Preparationmentioning

confidence: 99%

“…For example, Sadayappan and colleagues [86] used 1-D-IEF to investigate MyBP-C phosphorylation levels in several pathological states. To determine the phosphorylation states of TnI (pI 9.2), we developed a non-equilibrium IEF method (NEIEF) to resolve basic proteins [87], and used it to study TnI phosphorylation in TG mice expressing PKCe [20]. Usually, 1-D-IEF is followed by Western blot to visualize the separation of targeted proteins.…”

Section: Phosphorylationmentioning

confidence: 99%

Myofilament proteins: From cardiac disorders to proteomic changes

Yuan

Solaro

2008

Proteomics Clinical Apps

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Myofilament proteins of the cardiac sarcomere house the molecular machinery responsible for generating tension and pressure. Release of intracellular Ca 21 triggers myofilament tension generation and shortening, but the response to Ca 21 is modulated by changes in key regulatory proteins. We review how these proteomic changes are essential to adaptive physiological regulation of cardiac output and become maladaptive in cardiac disorders. We also review the essentials of proteomic techniques used to study myofilament protein changes, including degradation, isoform expression, phosphorylation and oxidation. Selected proteomic studies illustrate the applications of these approaches.

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Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCε phosphorylation

Cited by 58 publications

References 31 publications

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

Analytical strategies for phosphoproteomics

Myofilament proteins: From cardiac disorders to proteomic changes

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