☆ 4.5 Article

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force

BIOPHYSICAL JOURNAL (2015)

Journal

BIOPHYSICAL JOURNAL

Volume 109, Issue 12, Pages 2592-2601

Publisher

CELL PRESS

DOI: 10.1016/j.bpj.2015.11.002

Keywords

Funding

National Institutes of Health [HL062881, HL115988]
European Union grant EU-FP7
Foundation Leducq
Transatlantic Network of Excellence [14CVD03, 13CVD04]

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Abstract

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKII delta) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKII delta and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (similar to 30%) passive force reduction caused by CaMKII delta and a much smaller (similar to 6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKII delta can alter the passive force of myocytes in the heart (although CaMKII delta appears to be more potent) during physiological and pathophysiological states.

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Preparing Excitable Cardiac Papillary Muscle and Cardiac Slices for Functional Analyses

Bradley M. Palmer, Stephen P. Bell

Summary: The reductionist approach has been valuable in understanding muscle function at the molecular level, but intact excitable muscle preparations still play a crucial role as experimental model systems. This article presents methods for preparing cardiac papillary muscle and cardiac slices, which serve as macroscopic experimental models with fully intact intercellular and intracellular structures. These model systems have proven to be especially useful for testing the functional effects of protein mutations and pharmaceutical candidates. The article provides solutions recipes for dissection and recording, as well as instructions for removing and preparing the cardiac papillary muscles and cardiac slices. These instructions are suitable for novice experimentalists and can also serve as a refresher for experienced muscle physiologists.

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