Does β‐Adrenergic/PKA Signaling Modulate Ca 2+ ‐Sensitivity and Force Kinetics in Myofibrils from Mice Carrying the FHC Related Mutation cTnI‐ΔK184?

FHC related mutations have been reported to interfere with myofibrillar responses to cAMP‐PKA signaling. We have previously shown that a deletion mutation in the C‐terminus of cTnI (cTnI‐ΔK184) increased Ca 2+ ‐sensitivity, slowed relaxation and increased passive stiffness of cardiac myofibrils (Mf) isolated from transgenic mice (Tg) carrying this mutation. The impaired relaxation, which likely underlies diastolic dysfunction, could be ascribed to incomplete inactivation of crossbridges at resting pCa (Iorga et al., 2007). Here, we tested the hypothesis that these biomechanical effects are modulated by β‐adrenoceptor mediated phosphorylation of sarcomeric proteins. To alter phosphorylation in vivo , Tg and WT mice were treated with i.p. injection of propranolol (30 mg/kg b.w.) 1 hr before sacrifice (propranolol treated, PT) in accordance with the Directive 2010/63/EU and German Animal Welfare Act. Skinned fibers (Sf) and Mf were isolated from hearts of such treated mice and untreated control mice and analyzed for mechanical performance. We further tested whether in vitro treatment of these preparations with PKA reversed the PT mediated effects on biomechanics. ProQ Diamond staining and cTnI‐pS22/pS23 phosphospecific antibodies detected no significant difference in cTnI and MyBP‐C phosphorylation between Tg and WT untreated mice. In such untreated mice, Ca 2+ ‐sensitivity of Tg‐Sf from was higher compared to WT (pCa 50 ‐values: WT 5.48, Tg 5.65, p<0.01). PT significantly lowered phosphorylation in both lines by a similar extent which was associated with a right‐ward shift in the force‐pCa relation in Tg‐Sf by 0.07 units but unexpectedly not in WT‐Sf. While PKA had no effect on the force‐pCa relation in Sf from untreated mice, it decreased Ca 2+ ‐ sensitivity by ~ 0.11 pCa‐units in WT‐Sf and ~0.21 pCa‐units in Tg‐Sf from PT mice. The changes in Ca 2+ ‐sensitivity were associated with respective changes in cTnI phosphorylation. The passive force‐sarcomere length (SL) relation was shifted upward in WT‐Mf from PT but surprisingly, this effect was not reversed by PKA. In contrast, PT had no effect on this relation in Tg‐Mf but subsequent incubation with PKA shifted it downward. PT slowed relaxation kinetics only in WT and treatment with PKA only reversed the slowing effect on the duration of the initial phase of relaxation. However, we cannot exclude possible confounding effects of PKA buffer. Neither PT nor PKA altered relaxation kinetics in Tg‐Mf. In summary, our results suggest that propranolol modulates phosphorylation sites in addition to PKA sites, which appear to manifest differently in WT and Tg. Still, our results indicate that the Ca 2+ ‐desensitizing effect of PKA is not abrogated by the FHC related cTnI‐D184 mutation. Importantly, propranolol slows relaxation kinetics in WT but not in Tg suggesting that propranolol does not worsen the diastolic dysfunction. Support or Funding Information German Research Foundation (SFB 612 to GP and RS). DM was supported by the graduate program in Pharmacology and Experimental Therapeutics at the University of Cologne which is financially and scientifically supported by Bayer and Medical Faculty of the Universty of Cologne