Identification of a direct interaction of protein kinase A with the L-type Ca2+ channel and characterization of effects of PDE3A mutations on cardiac myocytes

Abstract

In the heart, the excitation-contraction coupling (ECC) pathway links excitation at the plasma membrane to contraction of the cardiomyocytes. The L-type Ca2+ channel (LTCC) and phosphodiesterase 3A (PDE3A) play important roles in this pathway by facilitating Ca2+ influx into the cytosol and hydrolysis of cAMP at subcellular compartments e.g. the sarcoplasmic reticulum (SR) and in the cytosol. Regulation of the LTCC opening is modulated by proteins found in its local environment, amongst them the catalytic subunit of PKA (PKA-CS). However, how PKA-CS is tethered to the channel is unclear. Recently, it was found that the GTP-binding protein Rad is associated with the LTCC and is blocking the pore-forming a1c subunit in the basal state (Liu et al., 2020). PKA phosphorylation of Rad in response to a-adrenergic stimulation leads to its dissociation from the channel complex, releasing the inhibitory effect. In the first part of this work, a novel interaction of PKA-CS with the C terminus of the a1c subunit is described. This interaction was mapped to two regulatory regions of a1c, the distal and the proximal regulatory domain (DCRD and PCRD). Mutating the DCRD and PCRD regions affected the interaction with PKA-CS in vitro but not in a cellular environment, pointing towards a complex regulatory mechanism involving several proteins or an alternative recruitment process. The two regions did not affect the enzymatic activity of PKA-CS. The LTCC and isoform 1 of PDE3A were found in a complex in HEK293 cells, however no further functional link could be found. In the second part of this thesis, effects of mutations in PDE3A were characterized. These mutations cause hypertension with brachydactyly type E (HTNB). Affected individuals experience progressive hypertension and have characteristic shorter metacarpals. If untreated, patients die before the age of 50. Surprisingly, their hearts do not show hypertension-induced cardiac damage. This points to a cardioprotective effect of PDE3A mutations. HiPSC-CMs expressing T445N or R862C PDE3A HTNB-causing substitutions were established and differentiated to cardiac myocytes as a model system to study Ca2+ cycling. The mutant cells showed lower PDE3A and the LTCC a1c subunit protein levels and a longer Ca2+ dwell time in the cytosol compared to the wild-type (WT) cells. This might be an adaptive mechanism to improve contractility of the cells that contributes to the cardioprotective effect of the mutations.