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Diastolic performance is regulated by net myocardial stiffness, which is determined by the mechano-sensitive protein network comprised of extracellular proteins such as collagen, intracellular sarcomeric proteins, and cell surface integrins. Mechanical force and its regulation are sensed and propagated by each of these components in a concerted fashion.1 During diastole, titin filaments serve as tensiometers and passive force generators. This is accomplished by modulation via directional signaling of multiple linkages between different regions within the titin molecule and the cellular contractile apparatus. An intricate network of signaling molecules coordinates the extracellular and intracellular components in the contraction of a sarcomere. In this issue of Circulation Research, Hidalgo and colleagues present an elegant set of experiments that reveal a novel mechanism whereby altering the phosphorylation state of titin modulates myocardial passive stiffness.2 Specifically, they demonstrated that titin, in addition to being a protein kinase A and protein kinase G substrate, is also a protein kinase C α (PKCα) substrate. They further identified the PEVK region of titin as the prominent site of PKCα phosphorylation, and showed that phosphorylation at this site increased passive tension.
Titin is an approximately 3,000 kD molecular weight sarcomeric protein that spans the Z disk to the M line of the sarcomere.3 Originally thought to only provide structural scaffolding to link the many regulatory, contractile, and structural proteins within the sarcomere, titin is now recognized to be a major regulator of intracellular myocyte stiffness. Titin determines the passive tension of the intracellular component of cardiomyocytes, which together with collagen determines the total myocardial passive stiffness. While the immunoglobulin-like domain and fibronectin type III repeats make up 90% of the titin molecule, titin also includes a unique I-band region that is flexible and specifically serves as a molecular spring to determine passive stiffness. The I-band region contains 3 motifs: a) serially-linked immunoglobulin-like domains; b) the N2B element; and c) the PEVK region. Titin has direct and indirect links with many signaling molecules and has multiple phosphorylation sites in the Z disk, M band, and I-band segments, which establishes titin as a major regulator of myocyte signaling.1 For example, the N2B element has previously been shown to be phosphorylated by protein kinase A and G to decrease stiffness.4, 5 This study extends these observations to demonstrate that the PEVK region can be phosphorylated by protein kinase Cα.
There are three titin isoforms, including a fetal form and two adult forms (N2B and N2BA), that all arise from differential splicing of one gene to result in proteins of different length I bands. The adult N2B is the shortest form and results in the highest stiffness; the N2BA is longer and provides a medium level of stiffness; and the N2BA fetal form is the longest and most compliant. The difference in sizes among the isoforms is due to increased immunoglobulin G and PEVK sites and the presence or absence of N2A site within the I band. As sarcomeres are stretched beyond the slack length, passive force is developed. Stretching the adult N2BA titin within a physiological range of 1.8-2.4 μm straightens out the folded Ig domain but does not significantly extend the PEVK and N2B regions, which keeps passive force low.1 Phosphatase activity increases the phosphorylation-directed decrease in stiffness, indicating that titin is phosphorylated in the basal state to maintain homeostatic stiffness. When the shorter N2B titin is stretched to the same sarcomere length, strain on the I band is much greater and the PEVK and N2B regions elongate, which sharply raises the passive force. Radke and colleagues have demonstrated that targeted deletion of only the N2B region increased the extension of the remaining elements and resulted in diastolic dysfunction.6 Interestingly, PKCα phosphorylated two sites in the PEVK region, but no sites in the N2B or Ig1-8 elements were found to be phosphorylated. Also, because a shift in isoforms from N2B to N2BA would result in more PEVK sites available for phosphorylation, thus isoform shifting provides a trigger mechanism to amplify the signaling potential.
Multiple studies have shown that the ratio between N2B and N2BA determines the titin contribution to passive stiffness, with increased N2B/N2BA ratios resulting in increased stiffness. The ratio can be changed by changing hemodynamics, which can induce isoform shifts. The Granzier laboratory has previously shown that, in a canine rapid pacing model of 4 weeks duration, the N2B level increased and the N2BA level decreased to raise the ratio.7 Similarly, hypertensive rats also show an increase in the N2B to N2BA ratio.8 In these cases, the increase in ratio was associated with increase in stiffness. On the contrary, patients with coronary artery disease or dilated cardiomyopathy show an increase in N2BA levels, which lowers the ratio. Patients with dilated cardiomyopathy who had the lowest passive myocardial stiffness showed the highest N2BA levels but the lowest amount of diastolic dysfunction.9 However, another study documented that patients with congestive heart failure showed hypophosphorylation of titin and increased stiffness.10 Stimulation of the β adrenergic receptor increases protein kinase A levels, which in turn would decrease titin stiffness. Whether these patients were on beta blockers to explain the difference between animal studies and human trials needs to be investigated to determine the consequence of β-adrenergic inhibition on LV diastolic function in patients with diastolic heart failure. These data suggest that isoform switching is one mechanism, but may not be the only mechanism, whereby titin regulates myocardial passive tension.
Isoform switching is a longer term mechanism, whereas phosphorylation provides a rapid mechanism to change tension. Figure 1 illustrates the putative mechanism of modulating passive tension via the phosphorylation and the isoform switching pathways. In support of this complex mechanism the current study provides novel insights into a more rapid and transient way for titin to regulate stiffness. The Granzier laboratory has previously investigated the relationship between long-term hypothyroidism and titin isoform levels.3 The fetal cardiac titin isoform is longer, with additional PEVK and Ig domain exons. While the previous study attributed the decrease in tension to an increase in the fetal isoform, this study extends previous observations to suggest that additional PEVK sites may provide additional phosphorylation sites.
While this study reveals new insights into titin regulation, several questions remain to be answered. For one, is the phosphorylation specific to PKCα or, as suggested by the authors, do other PKC isoforms also phosphorylate the PEVK region of titin? In addition, while the PEVK region had two dominant phosphorylation sites that result in increased stiffness, the possibility that other PEVK sites are phosphorylated and may increase or decrease stiffness to have functional significance needs to be explored. What downstream signaling molecules are differentially regulated by different titin isoforms or phosphorylation states also remains to be evaluated. Further, how exactly does the intracellular environment coordinate with the extracellular matrix? Several studies have shown that passive force can decrease at the single cardiomyocyte level, but overall LV diastolic wall stiffness increases due to a net increase in collagen deposition.11 This suggests that titin may be trying to provide a compensatory mechanism to decrease overall LV wall stiffness. When ECM levels are kept low, the change in titin isoforms and phosphorylation state provide sufficient compensation to keep LV wall stress low. Titin and collagen interaction have not been fully delineated. Specifically, we need more details on how this crosstalk is regulated in the physiological and pathological states. Furthermore, explorations into the therapeutic potential of altering titin isoforms and/or their phosphorylation states via the alpha and beta adrenergic pathways are warranted, since stimulation of the two pathways results in disparate outcomes (Figure 1).
In summary, the study by the Granzier laboratory extends the results of several previous publications to highlight the several mechanisms whereby titin can be regulated to control myocardial passive stiffness and regulate the complex signaling cascade of LV hypertrophy and increased stiffness. While titin function was initially thought to be restricted to scaffolding sarcomeric proteins and providing myofibrillar elasticity, titin is now known to serve prominent roles as a stress-sensor and regulatory upstream signaling molecule to modulate cardiac muscle gene expression during hypertrophy. Future studies will need to delineate the full complement of mechanisms that regulate titin function and the downstream consequences of titin function, in order to determine if inhibiting phosphorylation at specific titin sites would result in a positive outcome in the setting of diastolic dysfunction and heart failure.
Sources of Funding
The authors acknowledge grant support from NIH R01 HL75360, AHA Grant-in-Aid 0855119F, the Morrison Fund, and the Max and Minnie Tomerlin Voelcker Fund (to MLL).