Hyperphosphorylation of Mouse Cardiac Titin Contributes to Transverse Aortic Constriction-Induced Diastolic Dysfunction

Abstract

Rationale Mechanisms underlying diastolic dysfunction need to be better understood. Objective To study the role of titin in diastolic dysfunction using a mouse model of experimental heart failure induced by transverse aortic constriction (TAC). Methods and Results Eight weeks post-TAC surgery mice were divided into heart failure (HF) and congestive heart failure (CHF) groups. Mechanical studies on skinned LV myocardium measured total and titin- and extracellular matrix (ECM)-based passive stiffness. Total passive stiffness was increased in both HF and CHF mice and this was due to increases in both ECM-and titin-based passive stiffness, with titin being dominant. Protein expression and titin exon microarray analysis revealed increased expression of the more compliant N2BA isoform at the expense of the stiff N2B isoform in HF and CHF mice. These changes are predicted to lower titin-based stiffness. Because titin’s stiffness is also sensitive to titin phosphorylation by protein kinase A (PKA) and protein kinase C (PKC), back phosphorylation and Western Blot assays with novel phospho-specific antibodies were performed. HF and CHF mice showed hyperphosphorylation of PKA sites and the PEVK S26 PKC sites, but hypophosphorylation of the PEVK S170 PKC site. Protein phosphatase I (PP1) abolished differences in phosphorylation levels and normalized titin-based passive stiffness levels between Ctrl and HF myocardium. Conclusion TAC-induced HF results in increased ECM-and titin-based passive stiffness. Changes in titin splicing take place, which lower passive stiffness, but this effect is offset by hyperphosphorylation of residues in titin spring elements, particularly of PEVK S26. Thus complex changes in titin take place that combined are a major factor in the increased passive myocardial stiffness in HF.

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“…125 In experimental mouse hearts exposed to transverse aortic constriction surgery, PEVK site S11878 (PKCα) was hyper-phosphorylated, but PEVK site S12022 (CaMKIIδ/ PKCα) was hypo-phosphorylated. 114 These findings suggest that differential phosphorylation of elastic titin elements is common to failing hearts. Because phosphorylation of N2-Bus and PEVK can have opposite effects on cardiomyocyte passive tension, it is important to determine site-specific rather than total phosphorylation of titin in HF.…”

“…In the hearts of mice exposed to transverse aortic constriction, the N2BA:N2B ratio was significantly increased in comparison with healthy mouse hearts. 114 Relatively more N2BA than in healthy hearts was also observed in rat hearts with chronic ischemic cardiomyopathy because of a ligation of the left anterior descending coronary artery (LAD model). 105 Likewise, a hypothyroid rat model showed elevated cardiac N2BA:N2B expression ratios compared with normothyroid rat hearts.…”

Renin overexpression leads to increased titin-based stiffness contributing to diastolic dysfunction in hypertensive mRen2 rats

“…We found increased phosphorylation at Ser-12742, without changes in the phosphorylation level of Ser-12884 in mRen2 hearts. PEVK Ser-12742 hyperphosphorylation was previously demonstrated in mice with transverse aortic constriction (22), in an old HTN dog model (18), as well as in human HF (17,26). Moreover, a recent translational work implicated the hyperphosphorylation of the very same site (Ser-12742) in human HFpEF (45).…”

“…Beyond that, Western immunoblotting was applied to assess titin phosphorylation at particular serine (Ser) residues of PEVK segment (rich in proline, glutamate, valine and lysine amino acids) (30). The phospho-specific antibodies were validated by Granzier et al (20,22). Phospho-specific antibodies were developed against Ser-11878 (PS26; GL Biochem, Shanghai, China; 1:1,000) and Ser-12022 (PS170; Genscript, Piscataway, NJ; 1:1,000) in the full human sequence.…”

“…Previous reports suggested that the PEVK domain cannot be detected by Pro-Q Diamond stain (22). Accordingly, the PEVK phosphorylation was tested here with phospho-specific antibodies against phosphorylated Ser-12742 and Ser-12884 residues.…”