PKC activation reduces contraction force in human and rabbit myocardium by decreasing Ca2+ sensitivity

Background

Protein kinase C (PKC) activity is increased in heart failure and cardiac remodeling. It regulates numerous downstream signals and can directly phosphorylate troponins. Hence, PKC impacts several biological functions in cardiomyocytes, including their contractility and Ca²⁺ sensitivity. However, animal studies focusing on PKC report controversial results, e.g., regarding its effect on contractility. Therefore, this study aims at unraveling the functional and downstream signaling effects of PKC activation via the Diacylglycerol (DAG) mimetic phorbol-12-myristat-13-acetat (PMA) in beating myocardial slices from healthy rabbit and failing human hearts. 

Methods

Contraction forces (CF) of left ventricular human (n=6-8/4; technical/biological replicates) and rabbit (61-66/11) myocardial slices cultured under permanent (0.5 Hz) electrical stimulation with/out 24 h PMA treatment (human: 10-50 nM after 9-15d; rabbit: 50 nM at 2d) were measured. Rabbit slices (n=4-13/3-5) were exposed to transcription (2 mM actinomycin D), translation (20 mg/ml cycloheximide), and pan PKC (250 nM sotrastaurin + 50 nM staurosporin) inhibitors for 2h, followed by additional PMA treatment (24h). PKC-specific phosphorylations of PKD (Protein kinase D) at ser744-748, troponin I (TPNI) at S23/24, and T143 were analyzed via Western blotting after 30 min, 90 min, 150 min and 24 h PMA treatment. Ca²⁺ sensitivity in rabbit slices (n=4-5/3; control vs PMA (24 h)) was assessed. Maximum contraction force (CF_max) (n=4-6/3) was tested by addition of 100 nM isoprenaline. Ca²⁺ signals (n=11/10) were measured after loading cardiac slices with Calbryte as Ca²⁺ indicator.

Results

PMA reduced CF of human (-80%±11.7%) and rabbit (-33%±14.4%) cardiac slices, while no changes were observed in controls (p<0.01). PMA enhanced phosphorylations of ser744-748 of PKD (2.2-fold) and T-143 of TPNI (4.4-fold) 24 h after treatment, however, no changes were observed at phosphorylation site S23/24. TPNI-T-143 phosphorylation was already increased 30 min (1.5-fold) and 90 min (2.4-fold) after PMA addition. Actinomycin D and cycloheximide did not alter PMA-induced effects on CF and TPNI-T-143 phosphorylation. Pre-treatment of rabbit cardiac slices with sotrastaurin and staurosporin prevented the decrease in contractility, as otherwise caused by single PMA treatment. Initial results indicate that the phosphorylated amounts of ser744-748 of PKD and T-143 of TPNI were also reduced to control levels in these slices. PMA reduced Ca²⁺ sensitivity in rabbit slices by 0.16 pCa units (0.24 mM) (p<0.05), but neither F_cmax, nor the of Ca²⁺ signal amplitude.

Conclusions

PKC activation hampers contractility of rabbit and human myocardium, presumably via phosphorylation of TPNI-T-143, thereby, reducing Ca²⁺ sensitivity. This occurs also in the presence of transcription and translation inhibitors, suggesting a direct PKC-TPNI interaction not requiring gene expression.