Purpose: Impaired sarcoplasmic reticular (SR) Ca2+ uptake resulting from decreased SR Ca2+-ATPase type 2a (SERCA2a) expression or activity is characteristic of heart failure (HF) with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and/or activity as novel approaches to HF management. We here explore the role of Pak1 in maintaining ventricular Ca2+ homeostasis and electrophysiological stability under both physiological and.acute β-adrenergic or hypertrophic challenge conditions
Methods: Homozygous Pak1 flox (Pak1f/f) mice were used in control groups against mice expressing cardiac-specific knockout of Pak1 generated using the Cre-LoxP system whereby Cre is expressed under the α-myosin heavy chain promoter. Pak1cko. 8-10 week old male Pak1f/f and Pak1cko mice were subjected to either isoprenaline (ISO, Sigma) at 10 mg/kg/day or vehicle (saline) for 14 days via subcutaneously implanted osmotic mini-pumps (Alzet). Detailed characterisatiions by in vivo, ex vivo and single cell electrophysiological measurements, single cell [Ca2+]i measurements, molecular biological experiments were followed up.
Results: Mice with a cardiomyocyte-specific Pak1 deletion (Pak1cko), but not controls (Pak1f/f), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute β-adrenergic or hypertrophic challenge induced by isoproterenol. Isolated Pak1cko ventricular myocytes correspondingly showed aberrant cellular Ca2+ homeostasis. Pak1cko hearts showed an associated impairment of SERCA2a function and down-regulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demosntrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels following phenylephrine stress in cultured neonatal rat cardiomyocytes (NRCMs). This was abolished by the Pak1-knockdown in Ad-shPak1-infected NRCMs and increased by constitutive over-expression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor (SRF), a transcriptional factor well-known for its vital role in regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a.
Conclusions: These findings indicate that Pak1 is required to maintain ventricular Ca2+ homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.