Modulation of cardiac titin stiffness in diabetic and exercised hearts

Titin is the largest protein known to date and one of the main molecular components contributing to the passive stiffness of striated muscle tissue. In the mammalian heart, titin-based myofilament stiffness is an important determinant of myocardial distensibility, and diastolic and systolic function. The passive mechanical properties of cardiac titin largely depend on the expression ratio of the isoforms N2BA (~3.2 to 3.7 MDa, more compliant) and N2B (~3.0 MDa, stiffer). In contrast to cardiac muscle, skeletal muscle only expresses the N2A isoform (3.3-3.7 MDa). Titin-based passive tension can be dynamically modified by phosphorylation through the kinases PKA, PKG, PKCα, CaMKIIδ and ERK 1/2. Importantly, phosphorylation of titin's N2-Bus region by CaMKIIδ, PKA and PKG reduces titin-based myofilament stiffness, whereas phosphorylation of titin's PEVK region by PKCα increases it. This study investigated the effect of altered insulin availability on titin isoform composition and phosphorylation in human heart tissue sample from diabetes mellitus patients and insulin-treated embryonic and adult rat cardiomyocytes. The study further investigated altered titin stiffness and phosphorylation in adult rat cardiomyocytes induced by the oral anti-diabetic drug metformin. Results of the study at hand revealed the influence of altered insulin homeostasis on titin isoform ratio and titin phosphorylation by using samples from right atria of diabetic and non diabetic patients that underwent cardiac surgery due to coronary artery disease. Diabetic heart samples showed a significant increase in titin N2BA expression that was most pronounced in patients with high HbA1c levels (Glycated hemoglobin). Western blot analyses using phospho-specific antibodies showed a hypophosphorylation of titin at Ser4099 and Ser4010 within the N2-Bus and concomitantly a hyperphosphorylation of the PEVK element of titin at Ser11878. These phosphorylation changes are known to cause an increase in passive myocyte tension (PT). The PT of isolated cardiomyocytes from diabetic patients showed in fact a significant increase, suggesting a reduction in PKA and PKCα activity. Insulin-dependent changes in titin phosphorylation were further characterized using cultured embryonic and adult cardiomyocytes. Here, insulin and the oral anti-diabetic drug metformin caused a major increase in the relative phosphorylation at Ser4099 of titin's N2-Bus within 15 minutes of treatment. Furthermore, insulin treatment induced phosphorylation of the PKCα-sensitive site Ser11878 in the PEVK region of titin. Interestingly, PT was significantly increased in adult rat cardiomyocytes stimulated in vitro with metformin and insulin. Additionally, the effects of acute and chronic exercise (concentric and eccentric) on titin phosphorylation and myofilament stiffness in cardiac and skeletal tissue samples from physically trained rats were investigated. Therefore, adult rats were exercised acutely (15 minutes) and chronically (6 weeks) and non-exercised animals served as controls. PT from all exercised cardiac samples was significantly increased, compared to controls. In cardiac tissue titin N2-Bus phosphorylation was significantly decreased at Ser4099. However, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation was detected; however, PKCα activity remained unchanged. The evaluated phosphorylation status suggests a decrease in PT. However, the effect on skeletal muscle and PT still needs to be determined. In summary, the generated data show that insulin and metformin can be identified as potent modulators of cardiac titin and may therefore play an important role in modifying myocardial stiffness in human hearts. Furthermore, different kinds of exercise affect titin domain phosphorylation and titin-based cardiomyocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness caused by acute triggers may regulate adaptation processes of the passive and active properties of the myocardium.