Abstract 1953: Myocardial Titin Isoform Phosphorylation in Diastolic Heart Failure
Cardiomyocyte (CM) resting tension (RT) contributes to myocardial diastolic stiffness in patients with diastolic heart failure (DHF). The giant cytoskeletal protein titin modulates CM RT. CM RT falls when titin is phosphorylated by protein kinase A (PKA) and when the compliant N2BA titin isoform is overexpressed relative to the stiff N2B titin isoform. Especially phosphorylation of the stiff N2B titin isoform lowers RT. To explore involvement of titin isoform phosphorylation in the high RT of CM of DHF patients, expression and phosphorylation of titin isoforms and RT of isolated CM were determined in LV myocardial biopsies of DHF patients (n=16), of controls (CON) (n=10) and of patients with aortic stenosis (AS) (n=20). All patients were free of coronary artery disease and their biopsies showed no inflammatory infiltration. CM were treated with Triton X-100, attached to a force transducer and stretched to a sarcomere length of 2.2 microm to measure RT before and after administration of PKA. Expression and phosphorylation of titin isoforms were analysed using gel electrophoresis with SYPRO Ruby and Pro-Q Diamond Phosphoprotein Stain and reported as ratio of titin isoforms (N2BA/N2B) or as ratio of phosphorylated titin isoforms (P-N2BA/P-N2B). CM RT was higher in DHF (6.6±0.6 kN/m2; p<0.01) than in CON (3.7±0.5 kN/m2) or AS (2.4±0.2 kN/m2). PKA lowered CM RT in DHF (−2.6±0.5 kN/m2; p<0.01) but not in CON or in AS. Expression of titin isoforms (N2BA/N2B) was comparable in DHF (0.69±0.08), CON (0.45±0.02) and AS (0.61±0.07) but phosphorylation of titin isoforms (P-N2BA/P-N2B) differed between DHF (0.80±0.05; p<0.05) and CON (0.27±0.03) or AS (0.56±0.06). This implies more P-N2BA and less P-N2B in DHF than in CON or AS.
Conclusion: The higher CM RT in DHF than in CON or AS is related to titin isoform phosphorylation and not to titin isoform expression. In DHF, deficient phosphorylation of the stiff N2B titin isoform explains both the high CM RT and its correction by PKA.