Felodipine protects human atrial muscle from hypoxia-reoxygenation dysfunction: a force-frequency relationship study in an in vitro model of stunning.
OBJECTIVE
We aimed at investigating contractile changes after hypoxia-reoxygenation and dobutamine challenge in superfused human atrial pectinate muscle to see whether high versus low stimulation rate during hypoxia might account for outcome differences compatible with the definition of an in vitro model of myocardial stunning and whether pretreatment with the dihydropyridine Ca2+ entry blocker felodipine might afford protection.


METHODS
Human right atrial trabeculae obtained from adult patients were superfused in an organ bath with oxygenated (O2 content 16 ml/l) and modified (NaHCO3 25.7 mmol/l) Tyrode's solution at 37 degrees C. Dobutamine (1 nmol/l to 10 micromol/l) was superfused in 10 oxygenated preparations to select the optimal drug concentration to be used in another 22 which were randomized. Group (A) consisted of time-related controls (Tyrodes's solution for 225 min at cycle length (CL) 1600 ms and no dobutamine). There were two test groups, respectively: (B) low (1600 ms CL) and (C) high (400 ms CL) stimulation rate. After 60 min of stabilization, in groups B and C, hypoxic superfusion (O2 content 5 ml/l) lasted 60 min, then reoxygenation (60 min) and dobutamine challenge (1 micromol/l, 15 min) were performed. Analysis of variance for repeated measures with the Greenhouse-Geisser correction, and a repeated measures model with structured covariance (preparation mass, length, width and time-varying time to peak tension) matrices were used whereby grouping (G), time (T) and G x T interaction were weighted. Force-frequency relationship and post-pausal potentiation were studied after each phase. Electrophysiology, histomorphometry and electron microscopy were carried out (n=6). Felodipine (0.1 micromol/l, n=5) pretreatment (15 min before hypoxia) was given in parallel experiments.


RESULTS
Time-related controls showed approximately 10% per hour decrease of developed tension and the Paradise test provided approximately 80% of control values. In test groups (as compared to baseline values) contractility was decreased approximately 65% after hypoxia-reoxygenation and it increased approximately 25% after dobutamine (G, 0.0065<P<0.0155; T, P=0.00005; G x T, P=0.00005). High stimulation rate during hypoxia worsened hypoxia-reoxygenation contractile changes, whereas reversibility after dobutamine was less. In both B and C groups during hypoxia, contractility decreased quite rapidly, although by 10 min or so a plateau (approximately 50%) was reached in group B, whereas in group C contractility decreased to <20%. None of the covariates contributed significantly to predict the dependent variables investigated. Force-frequency relationship and post-pausal potentiation were repeatable, paralleled overall changes due to hypoxia, reoxygenation and dobutamine challenge and were useful to discriminate Ca2+-related diastolic processes thus helping index myocardial contractile reserve. Force-frequency relationship was negative at high stimulation rates, concomitant to an abrupt change of shape and duration of action potential with little time for Ca2+-related Ca2+ release and ensuing systolic processes. Felodipine pretreatment enabled an unblunted response to dobutamine. Histomorphometry showed an unexpected 'fibrotic core'. At electron microscopy, subendocardial and deep part of the same pectinate muscles showed identical degrees of degenerative lesions. Superfused samples showed, unexpectedly, less anoxic lesions than preparations fixed within 15 min from surgical explant, although lesions were higher than in samples fixed immediately after explant.


CONCLUSIONS
This might be a relevant model, whereby pharmacological or physical interventions are tested. Native human atrial trabeculae might be used without dissection and/or preservatives. If high stimulation rate during hypoxia is used the power of hypothesis testing is maximized. Future studies with this material will be easier and comparatively smaller series might be investigated. Felo
Iwashiro
K
K
Laboratory of Cardiovascular Pharmacology, University of Rome La Sapienza, Italy.
Criniti
A
A
Sinatra
R
R
Dawodu
A A
AA
d'Amati
G
G
Monti
F
F
Pannarale
L
L
Bernucci
P
P
Brancaccio
G L
GL
Vetuschi
A
A
Gaudio
E
E
Gallo
P
P
Puddu
P E
PE
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D002121:Calcium Channel Blockers / Q000008:administration & dosage*; D046508:Culture Techniques; D004280:Dobutamine / Q000494:pharmacology; D015736:Felodipine / Q000008:administration & dosage*; D005260:Female; D006325:Heart Atria / Q000187:drug effects* / Q000503:physiopathology; D006439:Hemodynamics / Q000187:drug effects; D006801:Humans; D000860:Hypoxia; D008297:Male; D008854:Microscopy, Electron; D008875:Middle Aged; D008955:Models, Cardiovascular; D009200:Myocardial Contraction / Q000187:drug effects* / Q000502:physiology; D015425:Myocardial Reperfusion / Q000379:methods; D017682:Myocardial Stunning / Q000473:pathology / Q000503:physiopathology*; D010865:Pilot Projects; D012518:Sarcomeres / Q000648:ultrastructure
D002121:Calcium Channel Blockers; D004280:Dobutamine; D015736:Felodipine