Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation.
Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Marc N
MN
Hirt
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Jasper
J
Boeddinghaus
Faculty of Medicine, National Heart & Lung Institute, Imperial College London, United Kingdom.
Alice
A
Mitchell
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Sebastian
S
Schaaf
Dermatology and Venereology Department and Clinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Christian
C
Börnchen
DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany; Department of General and Interventional Cardiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Christian
C
Müller
Max-Delbruck-Center for Molecular Medicine (MDC), Berlin, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Germany.
Herbert
H
Schulz
Max-Delbruck-Center for Molecular Medicine (MDC), Berlin, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Germany.
Norbert
N
Hubner
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Justus
J
Stenzig
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Andrea
A
Stoehr
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Christiane
C
Neuber
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Alexandra
A
Eder
Faculty of Medicine, National Heart & Lung Institute, Imperial College London, United Kingdom.
Pradeep K
PK
Luther
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Arne
A
Hansen
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany. Electronic address: t.eschenhagen@uke.de.
Thomas
T
Eschenhagen
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure; D018031:Connexin 43 / Q000378:metabolism; D003593:Cytoplasm / Q000502:physiology / Q000648:ultrastructure; D004558:Electric Stimulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D012518:Sarcomeres / Q000502:physiology / Q000648:ultrastructure; D046509:Tissue Culture Techniques / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods*
D015415:Biomarkers; D018031:Connexin 43; D007545:Isoproterenol; D002118:Calcium
2-photon microscopy; Cardiac tissue engineering; Cardiomyocyte maturation; Cardiomyocytes; Electrical stimulation; Induced pluripotent stem cells