Background:
High blood pressure represents the leading cause of cardiovascular disease worldwide. Hypertension with brachydactyly type E (HTNB), a subtype of high blood pressure, resembles essential hypertension and is characterized by increased proliferation rates of vascular smooth muscle cells (VSMCs). The disease results from gain-of-function phosphodiesterase (PDE)3A gene mutations. Unexpectedly, latest studies revealed that HTNB-patients showed no signs of cardiac hypertrophy and maintained normal cardiac function. This project aims to elucidate the mechanisms responsible for the cardioprotective effect of PDE3A. To this end, we generated and characterized two tissue-specific PDE3A knockout mouse models.
Methods:
PDE3A-floxed mice were bred either with cardiomyocyte-specific α-myosin heavy chain promoter driven Cre line (αMHC-Cre) or with smooth muscle cell-specific deleter mouse line (SM22-Cre) to generate cardiac myocyte-specific or smooth muscle cell-specific knockout of all PDE3A isoforms (PDE3A-CM-KO vs. PDE3A-VSMC-KO). Cell type-specific knockout of PDE3A was evaluated by immunoblotting and live cell imaging. Immunostaining confirmed isolation of VSMCs and classical biochemical PDE assay was utilized to study cellular PDE3A activity. Phosphorylation of main components of the excitation-contraction coupling was analyzed under basal and various stimulated conditions. Effects of PDE3A-KO on proliferation of cultured VSMCs were investigated by examining the phosphorylation of key cell cycle regulatory proteins. Single-cell contractility measurements using IonOptix device were conducted to analyze calcium re-uptake and sarcomere relaxation.
Results:
PDE3A protein expression in cardiomyocytes from PDE3A-CM-KO line was completely abolished, as was PDE3A expression in VSMCs from PDE3A-VSMC-KO mice. Live cell imaging using FRET-based biosensors confirmed the global and cell-type specific PDE3A-knockout and PDE assay showed decreased cellular PDE3A activity. Investigation of downstream effects of PDE3A-KO on cardiac calcium handling proteins revealed significantly increased phosphorylation of phospholamban in PDE3A-CM-KO line. VSMCs isolated from PDE3A-VSMC-KO mice exhibited significant increased phosphorylation of phospholamban, CREB and MKP-1, accompanied by lower proliferation rates of cultured VSMCs. Studies with IonOptix demonstrated faster Ca2+ reuptake in PDE3A-KO compared to wildtype.
Conclusion:
PDE3A-KO knockout mouse lines were evaluated and should allow more detail studies of the cardioprotective effect of HTNB-causing PDE3A mutations. This includes phenotypic characterization in cardiac stress models with and without PDE3A hyperactive mutant overexpression, live cell imaging studies to analyze their effects on local cAMP-signaling and assessments of contractility and Ca2+ homeostasis in the isolated cardiomyocytes.
