The impact of ovariectomy on cardiac excitation-contraction coupling is mediated through cAMP/PKA-dependent mechanisms

J Mol Cell Cardiol. 2017 Oct;111:51-60. doi: 10.1016/j.yjmcc.2017.07.118. Epub 2017 Aug 1.

Abstract

Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca2+ overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca2+ dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca2+ concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca2+ currents were similar in myocytes from sham and OVX mice but Ca2+ transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2μM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300μM IBMX), or an AC activator (10μM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca2+ homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50μM). Interestingly, selective inhibition of PDE4 increased Ca2+ current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca2+ release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca2+ dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.

Keywords: Adenylyl cyclase; Estrogen; Excitation-contraction coupling; Phosphodiesterase; Sex difference.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Adenylyl Cyclases / metabolism
  • Animals
  • Calcium Signaling / drug effects
  • Cell Size / drug effects
  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cyclic Nucleotide Phosphodiesterases, Type 4 / metabolism
  • Excitation Contraction Coupling* / drug effects
  • Female
  • Isoquinolines / pharmacology
  • Mice, Inbred C57BL
  • Models, Biological
  • Myocardial Contraction / drug effects
  • Myocardium / metabolism*
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Myocytes, Cardiac / pathology
  • Organ Size / drug effects
  • Ovariectomy*
  • Protein Kinase Inhibitors / pharmacology
  • Rolipram / pharmacology
  • Sarcoplasmic Reticulum / metabolism
  • Sulfonamides / pharmacology

Substances

  • Isoquinolines
  • Protein Kinase Inhibitors
  • Sulfonamides
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclic Nucleotide Phosphodiesterases, Type 4
  • Pde4a protein, mouse
  • Adenylyl Cyclases
  • Rolipram
  • N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide