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 (F
dev
)-muscle length (L) relationships and [Ca]
i
(fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm,
R
= [emission fluorescence
340
]/[emission fluorescence
380
]).
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 F
dev
and slowed relaxation for all muscle lengths examined (F
dev
@ 100% L
max
, WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm
−2
,
P
= 0.005; dF/dt
min
@ 100% L
max
, 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
R
systole
/
R
diaastole
, WT: 1.62±0.07, TG: 1.48±0.22; [Ca]
i
relaxation rate d
R
/dt
min
, 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.
Regulation of the intracellular calcium transient and force of contraction via muscarinic receptors in the mammalian cardiac muscle