Abstract 5403: Protein Kinase C-Induced Troponin I Phosphorylation Causes Changes in Cardiac Contractile Function but not the Intracellular Calcium Transient
Both intracellular calcium transients ([Ca]i) and myofilament properties determine cardiac muscle contractile force. Transgenic mouse models created to perturb specific myofilament proteins often cause a compensatory change in [Ca]i, which confounds the assessment of myofilament structure-function relationships. We have created a new transgenic mouse that has all three protein kinase C (PKC) phosphorylation sites on cardiac troponin I (cTnI) mutated to glutamic acid, rendering these sites constitutively pseudo-phosphorylated. Our goal was to determine the effects of this mutation on cardiac muscle contractile function and whether these effects would be concurrent with changes in the [Ca]i. Two sets of studies were conducted:
skinned muscle fiber experiments to characterize the steady-state force-pCa relationships at sarcomere lengths of 1.9 and 2.3 μm and
right ventricular papillary muscle experiments to characterize the peak developed force (Fdev)-muscle length (L) relationships and [Ca]i (fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm, R = [emission fluorescence340]/[emission fluorescence380]).
In skinned fibers, there was a significant decrease in maximally activated force (i.e., force at pCa 4.33) in transgenic mice (Wild-Type, WT (n = 7): 64.4± 8.0, Transgenic, TG (n = 6): 42.6±6.8 mN•mm−2, P = 0.004), without any changes in calcium sensitivity or cooperativity (Hill coefficient). In intact papillary muscles, TG mice showed a decrease in Fdev and slowed relaxation for all muscle lengths examined (Fdev @ 100% Lmax, WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm−2, P = 0.005; dF/dtmin @ 100% Lmax, WT: −136±32, TG: −74±38 mN•mm−2•s−1, P = 0.002). In contrast, [Ca]i was unaltered in TG mice at all muscle lengths examined ([Ca]i amplitude as quantified by Rsystole/Rdiaastole, WT: 1.62±0.07, TG: 1.48±0.22; [Ca]i relaxation rate dR/dtmin, WT: −96±37, TG: −64±30 s−1). Thus, PKC-induced TnI phosphorylation affects cardiac muscle contraction (reduced force magnitude and slowed relaxation) via changes in the myofilament properties (activation and/or crossbridge dynamics), and these contractile effects are not related to any changes in the intracellular calcium transient.