Constitutive BDNF/TrkB signaling is required for normal cardiac contraction and relaxation

Abstract

BDNF and its associated tropomyosin-related kinase receptor B (TrkB) nurture vessels and nerves serving the heart. However, the direct effect of BDNF/TrkB signaling on the myocardium is poorly understood. Here we report that cardiac-specific TrkB knockout mice (TrkB(-/-)) display impaired cardiac contraction and relaxation, showing that BDNF/TrkB signaling acts constitutively to sustain in vivo myocardial performance. BDNF enhances normal cardiomyocyte Ca(2+) cycling, contractility, and relaxation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Conversely, failing myocytes, which have increased truncated TrkB lacking tyrosine kinase activity and chronically activated CaMKII, are insensitive to BDNF. Thus, BDNF/TrkB signaling represents a previously unidentified pathway by which the peripheral nervous system directly and tonically influences myocardial function in parallel with β-adrenergic control. Deficits in this system are likely additional contributors to acute and chronic cardiac dysfunction.

Keywords: BDNF; CaMKII; TrkB receptor; cardiac contractility/relaxation; neurotrophins.

Fig. 1.

BDNF increases cardiomyocyte contractility and relaxation as a result of enhanced Ca²⁺ cycling, in a TrkB receptor-dependent manner. (A) TrkB protein expression is detected in isolated adult murine cardiac myocytes by Western blot (140 KD). TrkB is located on plasma membrane of cardiac myocytes by immunohistochemical study, using confocal microscopy. (B) Representative traces of sarcomere shortening and Ca²⁺ transients, with or without BDNF treatment. Incubating isolated murine cardiomyocytes with BDNF (20 nM) increases myocyte fractional shortening and whole-cell Ca²⁺ transients while accelerating myocyte relaxation; the latter is documented by decreased relaxation time (n = 19). (C) Representative confocal imaging of Ca²⁺ spark events in isolated rat adult cardiomyocytes: BDNF augments Ca²⁺ spark frequency in these cells. (D) Consistent with data in the mice, BDNF increases Ca²⁺ transients and SR Ca²⁺ fractional release in isolated rat adult cardiomyocytes without significantly affecting diastolic Ca²⁺ levels or SR Ca²⁺ load, measured by caffeine-induced total SR Ca²⁺ release. (E) Raw traces of BDNF’s effect on L-type Ca²⁺ channel activity measured in isolated guinea pig ventricular myocytes: BDNF enhances peak L-type Ca²⁺ channel activity from baseline (P < 0.05). (F) BDNF inotropy is abolished in cardiomyocytes isolated from TrkB^F616A mice with 1-NNMP1 pretreatment.

Fig. 2.

Constitutive BDNF/TrkB signaling is required for normal cardiac contraction and relaxation. (A) Representative pressure-volume loops obtained in WT littermate mice and in cardiac-specific TrkB^−/− mice. (B) Cardiac contractility and relaxation are impaired in TrkB^−/− mice, as indexed by dp/dt_max, dpdt/EDV, dpdt/ip, prerecruitable stroke work, Ees/Ea ratio, and Tau logistic, respectively.

Fig. 3.

β-adrenergic response is intact in TrkB^−/− mice. (A) Representative pressure-volume loops obtained before and after the infusion of the β1-β2 agonist isoproterenol (40 ng/kg/min). (B) Isoproterenol increases in vivo contractility (dP/dt_max and dPdt/IP) with the same magnitude in WT and TrkB^−/− mice, despite the lower basal contractile values found in the TrkB^−/− mice (n = 5 each group).

Fig. 4.

CaMKII is the major mediator of BDNF influence on myocardial mechanics. (A) BDNF induces CaMKII phosphorylation in adult murine cardiac myocytes (n = 3) and increases the phosphorylation of CaMKII-dependent sites on RyR (serine 2814) and PLN (threonine 17), respectively (n = 4); TrkB^−/− mice display significantly reduced levels of constitutive phosphorylation of CaMKII, as indexed by the P-CaMKII/T-CaMKII ratio (n = 4). (B) Representative traces of BDNF’s effect on isolated mouse cardiomyocytes in the absence and the presence of the CaMKII inhibitor KN93: Pretreating cells with KN93 prevents BDNF enhancement of myocyte contraction (n = 10).

Fig. 5.

BDNF-evoked enhancement of cardiac contractility is lost in failing cardiomyocytes that display increased truncated TrkB and altered CaMKII signaling/targets. (A) Myocytes isolated from G_αq mice are insensitive to BDNF (20 nM) (control cells = 19; Galphaq myocytes = 24): raw traces and cumulative data for sarcomere shortening and whole-cell Ca²⁺ transients. (B) The expression of full-length TrkB is unchanged in G_αq OE hearts; however, the truncated TrkB (Trk-T1) is markedly increased. (C) In G_αq OE mice hearts, CaMKII phosphorylation is constitutively up-regulated, as shown by the increased P-CaMKII/T-CaMKII ratio. The expression of SERCA2a is markedly decreased in G_αq hearts; this change is coupled to unchanged expression of total PLN, and to reduced levels of PLN phosphorylation levels at the T17 residue (*P < 0.05, **P < 0.01).