ABSTRACT
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
Subject(s)
Calcium-Binding Proteins , Heart Failure , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Heart , Mice , RNA, MessengerABSTRACT
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Calcium-Binding Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Animals , Binding Sites , Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinase Type 2/chemistry , Cells, Cultured , HEK293 Cells , Humans , Myocytes, Cardiac/metabolism , Protein Binding , Rats , Rats, WistarABSTRACT
AIMS: Regional heterogeneities in contraction contribute to heart failure with reduced ejection fraction (HFrEF). We aimed to determine whether regional changes in myocardial relaxation similarly contribute to diastolic dysfunction in post-infarction HFrEF, and to elucidate the underlying mechanisms. METHODS AND RESULTS: Using the magnetic resonance imaging phase-contrast technique, we examined local diastolic function in a rat model of post-infarction HFrEF. In comparison with sham-operated animals, post-infarction HFrEF rats exhibited reduced diastolic strain rate adjacent to the scar, but not in remote regions of the myocardium. Removal of Ca2+ within cardiomyocytes governs relaxation, and we indeed found that Ca2+ transients declined more slowly in cells isolated from the adjacent region. Resting Ca2+ levels in adjacent zone myocytes were also markedly elevated at high pacing rates. Impaired Ca2+ removal was attributed to a reduced rate of Ca2+ sequestration into the sarcoplasmic reticulum (SR), due to decreased local expression of the SR Ca2+ ATPase (SERCA). Wall stress was elevated in the adjacent region. Using ex vivo experiments with loaded papillary muscles, we demonstrated that high mechanical stress is directly linked to SERCA down-regulation and slowing of relaxation. Finally, we confirmed that regional diastolic dysfunction is also present in human HFrEF patients. Using echocardiographic speckle-tracking of patients enrolled in the LEAF trial, we found that in comparison with controls, post-infarction HFrEF subjects exhibited reduced diastolic train rate adjacent to the scar, but not in remote regions of the myocardium. CONCLUSION: Our data indicate that relaxation varies across the heart in post-infarction HFrEF. Regional diastolic dysfunction in this condition is linked to elevated wall stress adjacent to the infarction, resulting in down-regulation of SERCA, disrupted diastolic Ca2+ handling, and local slowing of relaxation.
Subject(s)
Calcium Signaling , Heart Failure/etiology , Myocardial Infarction/complications , Myocytes, Cardiac/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Ventricular Dysfunction, Left/etiology , Ventricular Function, Left , Ventricular Remodeling , Aged , Animals , Computer Simulation , Diastole , Disease Models, Animal , Fibrosis , Heart Failure/metabolism , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Kinetics , Male , Middle Aged , Models, Cardiovascular , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocytes, Cardiac/pathology , Randomized Controlled Trials as Topic , Rats, Wistar , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/pathology , Ventricular Dysfunction, Left/physiopathologyABSTRACT
The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm × 3 mm × 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.