The inotropic peptide βARKct improves βAR responsiveness in normal and failing cardiomyocytes through G(βγ)-mediated L-type calcium current disinhibition.
BACKGROUND
The G(βγ)-sequestering peptide β-adrenergic receptor kinase (βARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased β-adrenergic receptor (βAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by βARKct and its impact on G(βγ)-mediated signaling have yet to be fully elucidated.


OBJECTIVE
We sought to identify G(βγ)-regulated targets and signaling mechanisms conveying βARKct-mediated enhanced βAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes.


RESULTS
Assessing viral-based βARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that βARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on βAR stimulation. Mechanistically, βARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(βγ) subunits downstream of activated βARs. Despite improved βAR contractile responsiveness, βARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although βARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(βγ) protein and use-dependent LCC blocker, βARKct-expressing cardiomyocytes exhibit equal basal and βAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls.


CONCLUSIONS
Our study identifies a G(βγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on βAR as molecular target for the G(βγ)-sequestering peptide βARKct. Targeted interruption of this inhibitory signaling pathway by βARKct confers improved βAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. βARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to βAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Mirko
M
Völkers
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Christian
C
Weidenhammer
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Nicole
N
Herzog
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Gang
G
Qiu
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Kristin
K
Spaich
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Frederic V
FV
Wegner
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Karsten
K
Peppel
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Oliver J
OJ
Müller
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Stefanie
S
Schinkel
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Joseph E
JE
Rabinowitz
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Hans-Jorg
HJ
Hippe
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Henriette
H
Brinks
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Hugo A
HA
Katus
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Walter J
WJ
Koch
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Andrea D
AD
Eckhart
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Oliver
O
Friedrich
Center for Molecular and Translational Cardiology (M.V, C.W., N.H., K.S., P.M.), Department of Internal Medicine III (O.J.M, S.S., H.J.H., H.A.K.), Division of Cardiology, INF 350, University of Heidelberg, 69120 Heidelberg, Germany; Institute of Physiology and Pathophysiology (F.W., O.F.) Medical Biophysics, INF 326, University of Heidelberg, 69120 Heidelberg, Germany; George Zallie & Family Laboratory for Cardiovascular Gene Therapy (J.E.R., H.B., W.J.K.), Eugene Feiner Laboratory for Vascular Biology and Thrombosis (A.D.E.), Laboratory for Cardiac Stem Cell and Gene Therapy (G.Q., K.P., P.M.), Center for Translational Medicine, Thomas Jefferson University, 19107 Philadelphia, PA, USA.
Patrick
P
Most
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000378:metabolism*; D002470:Cell Survival / Q000235:genetics; D054769:G-Protein-Coupled Receptor Kinase 2; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods*; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D006352:Heart Ventricles / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics; D009200:Myocardial Contraction / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000235:genetics / Q000378:metabolism
C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type; D002316:Cardiotonic Agents; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010455:Peptides; D011494:Protein Kinases; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; D002118:Calcium