Contribution of SERCA2, RyR2, NCX1 and PMCA to excitation-contraction coupling in human cardiomyocytes – tackling an old question with modern technologies

• Applicants: Privatdozent Dr. Torsten Christ; Professor Dr. Thomas Eschenhagen
• Subject Area: Pharmacology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 532692509

The central role of calcium for heart contraction is established since the pioneering work of Sydney Ringer. Textbooks summarize the process (simplified) as follows: (1) Calcium ions enter the cardiomyocyte through L-type calcium channels (LTCC) that open in response to membrane depolarization; (2) the influx of calcium triggers the release of more calcium ions from the sarcoplasmic reticulum (SR) by opening of ryanodine receptors; (3) calcium binds to the thin filament-based troponin C, which initiates rearrangement of the troponin/tropomyosin complex and activation of myosin-actin interaction and systolic force generation. The removal of cytoplasmic calcium via the SR-calcium ATPase (SERCA2; energy-consuming) and the plasmalemmal sodium-calcium exchanger (NCX1; ATP-independent, but electrogenic) reverses the activation processes and causes relaxation in diastole. The plasmalemmal calcium ATPase (PMCA) is considered to play a minor role and is therefore absent from most schemes. For the system to work stably under steady state conditions, the amount of calcium leaving the cell must exactly equal the amount entering it and, vice versa, the amount being sequestered into the SR in diastole must equal the amount released by the SR. The system is under control by β-adrenergic (and other) receptors that lead to increased calcium influx and calcium re-uptake by the SR. As predicted by the model, pharmacological inhibition of LTCC with calcium channel blockers invariably decreases force of contraction and stops the heart from beating at sufficiently high concentrations. Thus, influx of calcium is absolutely required for cardiomyocytes to beat. Surprisingly, however, several well-performed studies over the past two decades indicate that neither pharmacological nor genetic inhibition of SERCA2 or NCX1 relevantly affects cardiac force of contraction. Similarly, deletion or pharmacological inhibition of the plasma membrane calcium ATPase 4 (PMCA4) has no effects on cardiac contractility. Preliminary own experiments confirm these perplexing findings and indicate that even the combined inhibition of all three systems is without major effects on the contractile force of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissue (EHT). Given the limitations of pharmacological interventions, more work with more definite methods is needed to solve the apparent conundrum. The present project uses hiPSC, EHT, CRISPR/Cas9 for gene deletion, AAV-mediated transfer of targeted, genetically encoded calcium sensors and pharmacological interventions to the role of SERCA2, NCX1, PMCA4 und RyR2 in excitation-contraction coupling under controlled conditions in a human heart muscle context.

• DFG Programme: Research Grants