The contributions of cardiac myosin binding protein C and troponin I phosphorylation to β-adrenergic enhancement of in vivo cardiac function

Abstract

β-adrenergic stimulation increases cardiac myosin binding protein C (MyBP-C) and troponin I phosphorylation to accelerate pressure development and relaxation in vivo, although their relative contributions remain unknown. Using a novel mouse model lacking protein kinase A-phosphorylatable troponin I (TnI) and MyBP-C, we examined in vivo haemodynamic function before and after infusion of the β-agonist dobutamine. Mice expressing phospho-ablated MyBP-C displayed cardiac hypertrophy and prevented full acceleration of pressure development and relaxation in response to dobutamine, whereas expression of phosphor-ablated TnI alone had little effect on the acceleration of contractile function in response to dobutamine. Our data demonstrate that MyBP-C phosphorylation is the principal mediator of the contractile response to increased β-agonist stimulation in vivo. These results help us understand why MyBP-C dephosphorylation in the failing heart contributes to contractile dysfunction and decreased adrenergic reserve in response to acute stress. β-adrenergic stimulation plays a critical role in accelerating ventricular contraction and speeding relaxation to match cardiac output to changing circulatory demands. Two key myofilaments proteins, troponin I (TnI) and myosin binding protein-C (MyBP-C), are phosphorylated following β-adrenergic stimulation; however, their relative contributions to the enhancement of in vivo cardiac contractility are unknown. To examine the roles of TnI and MyBP-C phosphorylation in β-adrenergic-mediated enhancement of cardiac function, transgenic (TG) mice expressing non-phosphorylatable TnI protein kinase A (PKA) residues (i.e. serine to alanine substitution at Ser23/24; TnI(PKA-)) were bred with mice expressing non-phosphorylatable MyBP-C PKA residues (i.e. serine to alanine substitution at Ser273, Ser282 and Ser302; MyBPC(PKA-)) to generate a novel mouse model expressing non-phosphorylatable PKA residues in TnI and MyBP-C (DBL(PKA-)). MyBP-C dephosphorylation produced cardiac hypertrophy and increased wall thickness in MyBPC(PKA-) and DBL(PKA-) mice, and in vivo echocardiography and pressure-volume catheterization studies revealed impaired systolic function and prolonged diastolic relaxation compared to wild-type and TnI(PKA-) mice. Infusion of the β-agonist dobutamine resulted in accelerated rates of pressure development and relaxation in all mice; however, MyBPC(PKA-) and DBL(PKA-) mice displayed a blunted contractile response compared to wild-type and TnI(PKA-) mice. Furthermore, unanaesthesized MyBPC(PKA-) and DBL(PKA-) mice displayed depressed maximum systolic pressure in response to dobutamine as measured using implantable telemetry devices. Taken together, our data show that MyBP-C phosphorylation is a critical modulator of the in vivo acceleration of pressure development and relaxation as a result of enhanced β-adrenergic stimulation, and reduced MyBP-C phosphorylation may underlie depressed adrenergic reserve in heart failure.

Figure 1. MyBP‐C and TnI phosphorylation in TG lines

A, representative western blots of WT, TnI^PKA−, MyBPC^PKA− and DBL^PKA− cardiac samples demonstrating TnI and/or MyBP‐C phospho‐ablation. PVDF membranes were probed with primary antibodies specific for TnI phosphorylation (Ser23/24), total TnI, MyBP‐C phosphorylation (Ser273, Ser282 or Ser302), total MyBP‐C or HSC70. B, representative Coomasie and Pro‐Q stained cardiac myofibrils from TG and WT lines that were incubated in the presence or absence of the catalytic subunit of PKA (see Methods). C, quantification of MyBP‐C, TnI and troponin T phosphorylation from cardiac myofibrils for each line with or without PKA incubation. The intensity of the Pro‐Q band was normalized to the Coomassie band intensity and non‐PKA treated WT myofibril protein phosphorylation was set as 1. Myofibrils were isolated from six hearts for each mouse line. D, representative western blots of WT, TnI^PKA−, MyBPC^PKA− and DBL^PKA− cardiac ventricular samples demonstrating PLB phosphorylation. PVDF membranes were probed with primary antibodies specific for PLB phosphorylation (Ser16) in the absence (–) or presence (+) of dobutamine administration. E, quantification of PLB phosphorylation from cardiac samples for each line with or without dobutamine administration. PLB phosphorylation was normalized to total PLB protein expression and non‐dobutamine treated WT PLB phosphorylation was set as 1. Ventricular samples were isolated from three or four hearts for each mouse line.

Figure 2. Analysis of cardiac morphology

Representative formalin fixed hearts from WT, TnI^PKA−, MyBPC^PKA− and DBL^PKA− mice. After formalin fixation, representative hearts were imaged (A) (scale bar = 1 mm) before being sectioned at the mid LV and imaged again (B) (scale bar = 1 mm). C, cross‐sections of the mid LV were stained with Masson's trichrome and imaged at 100× magnification (scale bar = 50 μm). D, summary data showing heart weight normalized to body weight for each line. Values are expressed as the mean ± SEM from four or five mice per line. *Significantly different from WT (P < 0.05). E, quantification of fibrosis from Masson's trichrome cardiac sections in (C). Values are expressed as the mean ± SEM and cardiac sections were analysed from four or five mice per line.

Figure 3. Representative P–V loops from TG lines

Representative P–V loops from WT, TnI^PKA−, MyBPC^PKA− and DBL^PKA‐ mice. Representative loops were averaged from at least 10 P–V loops before (solid trace) and after (dashed trace) dobutamine administration for each animal.

Figure 4. MyBP‐C phospho‐ablation slows early pressure decline

A, representative pressure traces showing peak pressure decline from peak pressure. MyBPC^PKA‐ and DBL^PKA− mice show a prolonged early pressure decline from peak pressure to dp/dt_min compared to WT and TnI^PKA− mice. B, MyBPC^PKA− and DBL^PKA− mice display a significantly prolonged time from peak pressure to dp/dt_min compared to WT and TnI^PKA− mice. C, the slope of the pressure trace from peak pressure to dp/dt_min was significantly shallower in MyBPC^PKA− and DBL^PKA− mice compared to WT and TnI^PKA− mice, suggesting slower pressure relaxation. Values are expressed as the mean ± SEM from seven to 10 mice per line. *Significantly different from WT (P < 0.05).

Figure 5. MyBP‐C phospho‐ablation blunts the adrenergic mediated enhancement of systolic function

A, early pressure development in WT and TnI^PKA− mice is accelerated by β‐adrenergic activation, although this acceleration is blunted in MyBPC^PKA‐ and DBL^PKA− mice. Black traces represent baseline LV developed pressure, whereas grey traces represent post‐dobutamine pressure development. MyBP‐C phospho‐ablation also blunts the β‐adrenergic mediated enhancement of the maximum rate of pressure development ( dp/dt_max) (B, maximum power development (C) and the peak ejection rate (PER, normalized to end diastolic volume) (D). All values in (B) to (D) are represented as the values at the peak dobutamine response minus baseline values. Values are expressed as the mean ± SEM from seven to 10 mice per line. *Significantly different from WT (P < 0.05).

Figure 6. MyBP‐C phospho‐ablation blunts the adrenergic mediated enhancement of diastolic relaxation

A, ventricular pressure relaxation in WT and TnI^PKA− mice is accelerated by β‐adrenergic activation, although this acceleration is blunted in MyBPC^PKA‐ and DBL^PKA− mice. Black traces represent baseline LV developed pressure during diastolic relaxation, whereas grey traces represent post‐dobutamine pressure relaxation. MyBP‐C phospho‐ablation also blunts the β‐adrenergic mediated acceleration of the relaxation time constant (Tau, B), the decrease in the slope of the pressure trace from 50% to 25% of maximal pressure (C) and the peak filling rate (PFR, normalized to end diastolic volume) (D). All values in (B) to (D) are represented as the values at the peak dobutamine response minus baseline values. Values are expressed as the mean ± SEM from seven to 10 mice per line. *Significantly different from WT (P < 0.05).

Figure 7. MyBP‐C phospho‐ablation alters the timing of the cardiac cycle after β‐adrenergic stimulation

A, in WT and TnI^PKA− mice, dobutamine administration shortens the relative duration of IVC, ejection and IVR, and lengthens the relative duration of diastolic filling, whereas MyBPC^PKA‐ and DBL^PKA− mice displayed an increased relative duration of ejection time, a decreased relative duration of filling time and an unchanged IVC duration after dobutamine. The relative duration of IVR was increased by dobutamine in DBL^PKA− mice but unchanged in MyBPC^PKA− mice. All durations were normalized to the total length of the cardiac cycle and are presented as the percentage change from baseline values. B, dobutamine administration significantly shortened the relative duration of systole and prolonged the relative duration of diastole in WT and TnI^PKA− mice compared to baseline, whereas systole was relatively prolonged and diastole relatively shortened in MyBPC^PKA‐ and DBL^PKA− mice after dobutamine. All durations were normalized to the total length of the cardiac cycle and all values are presented as a percentage of baseline. *Significantly different from WT values (P < 0.05).

Figure 8. MyBP‐C phospho‐ablation disrupts in vivo systolic pressure development after β‐adrenergic stimulation

A, β‐adrenergic stimulation by dobutamine administration decreases maximum systolic pressure development to accommodate an increased HR (see WT and TnI^PKA−). MyBP‐C phospho‐ablation, however, results in significantly reduced systolic blood pressure after β‐adrenergic stimulation because of an inability to fully accelerate pressure development in the absence of MyBP‐C phosphorylation. SBP, systolic blood pressure. B, time to peak systolic blood pressure was significantly decreased by dobutamine administration in WT and TnI^PKA− mice but was not significantly shortened from baseline in MyBPC^PKA‐ and DBL^PKA− mice and was significantly longer (after dobutamine) compared to WT. *Significantly different from the corresponding baseline group (without dobutamine treatment) (P < 0.05). ^†Significantly different from WT under the same treatment (i.e. post‐dobutamine) (P < 0.05).