Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β

Abstract

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HF dys ) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HF dys ; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HF dys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HF dys and reactivated by CRT. Mass spectrometry of myofilament proteins from HF dys animals incubated with GSK-3β confirmed GSK-3β-dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HF dys , but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HF dys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.

Cellular and Molecular Aspects of Dyssynchrony and Resynchronization

“…A similar reduction in myocyte size has been reported in humans after receiving CRT [76]. However, if F max is not normalized to cell cross-sectional area, the force produced in all three groups (healthy, dyssynchronous heart failure, CRT) is similar [68], despite the middle group having much larger cells. This could imply that despite having more myofibers, these are in some way defective.…”

“…This is opposite that observed with synchronous heart failure, which increases calcium sensitivity in humans [69] and canines [68,70], changes thought due to a decline of PKA-phosphorylation of cardiac troponin I (TnI) at serines 22 and 23 [71]. This over-sensitization can result in diastolic dysfunction [72] and arrhythmias [73].…”

“…To determine this mechanism, we first explored how CRT impacts the myofilament, and found it fully recovers calcium sensitivity ( Figure 4B) to normal/healthy levels [74,75]. The mechanism of improvement involved enhanced phosphorylation of a myofilament protein(s), and from proteomic analysis, a candidate kinase that could explain this was shown to be glycogen synthase kinase 3β [68], GSK-3β ( Figure 4A, 4B). This kinase acted on several proteins in the M-band and Z-disk, regions considered essential for structural scaffolding of the myofilament.…”

“…Instead, the dominant mechanism was transient de-activation [66], where stretching the activated muscle led to cross-bridge detachment [67]. Thus, acute electro-mechanical de-coupling alters myofilament properties.Chronic dyssynchrony further changes myofilament function, depressing the responsiveness of sarcomere force development to calcium stimulation ( Figure 4A) [68]. This is opposite that observed with synchronous heart failure, which increases calcium sensitivity in humans [69] and canines [68,70], changes thought due to a decline of PKA-phosphorylation of cardiac troponin I (TnI) at serines 22 and 23 [71].…”

“…CRT reverses both phenotypes, reducing desmin oligomers and re-associating desmin to the Zdisk [78]. Importantly, desmin cleavage is mediated by its phosphorylation by GSK-3β, the same kinase involved in regulated Z-disk myofilament calcium sensitivity [68]. …”