7.On-Line Supplement
Isolation of Cardiomyocytes
Cardiomyocytes were isolated from Guinea pig hearts, using as an initial step Langendorff-perfusion with enzyme-containing solution, as described elsewhere [1]. After tissue digestion, right and left ventricles (RV, LV; respectively) were harvested and placed in separate Petri-dishes filled with cardioplegic solution (containing [in mM]: NaCl 4.0; KCl 10.0; K-glutamate 130.0; MgCl2 1.0; HEPES 5.0; Glucose 11.0; and CaCl20.025; pH 7.2).For RVbasecell isolation, roughly one quarter of the tissue closest to the atrio-ventricular boundary was used, and for RVapex the quarter that was furthest away. LVbase and LVapex tissue were dissected as for the RV, but a thin section was collected from either surface, to serve as a source for sub-epicardial and sub-endocardial cells. The remaining mid-myocardial layer was discarded.
All harvested tissue blocks were then minced into small pieces and gently agitated for separation of the cells. Cells were harvested by filtering the cell suspension with a mesh (250μm, Cadish Precision Meshes, London, UK). The procedure of agitation and filtering was repeated 2 to 3 times. Cell-containing suspensions for each of the source regions were centrifuged for 1min at 16g(PK121R; ALC, Cologno Monzese, Italy); the supernatant was discarded and the cell pellet carefully re-suspended for storage in normal Tyrode (containing [in mM] NaCl 140.0; KCl 5.4; MgCl2 1.0; HEPES 5.0; Glucose 11.0; CaCl2 1.8; pH 7.4), additionally including 1mgmL-1 bovine serum albumin and 17gmL-1 trypsin inhibitor to terminate any further enzyme activity.
Cell cross-sectional area (Sc) was not directly measured, but calculated with the formula Sc = (wc2)/12, assuming an ellipsoidal cross-section with a short-to-long axis ratio of 1:3[2], and then used for force normalisation.
Cell and Sarcomere Active Mechanical Properties at Slack Length
Tables A1 and A2 give a more detailed overview about region-specific mechanical properties of cells held at slack length. Sarcomere data from regionally isolated cells at slack length are shown in Tables A3 and A4 (see also Table 1 of the paper).
Table A1: Overview of regional differences in force development measured at slack length.
Region / %dL[% EDL0] / End-systolic force [mN/mm2] / dF/dtMAX [N/(ms*mm2)] / dF/dtMIN[N/(ms*mm2)]
RVbase / 5.08±0.69 (13) / 2.71±0.52 (13) / 60.0±13.1 (13) / 52.6±11.7 (13)
RVapex / 4.56±0.44 (14) / 3.11±0.37 (14) a b / 72.4±8.7 (14) a b c / 61.8±8.1 (14) a b
RV (all) / 4.81±0.40 (27) / 2.92±0.31 (27)d / 66.4±7.7 (27) d / 57.4±7.0 (27) d
LVbase_epi / 4.02±0.35 (16) / 1.84±0.17 (16) / 40.7±3.3 (16) / 34.7±3.4 (16)
LVbase_endo / 4.50±0.35 (19) / 2.19±0.27 (19) / 44.9±5.3 (19) / 43.5±4.6 (19)
LVapex_epi / 4.23±0.40 (16) / 2.36±0.36 (16) / 47.6±6.4 (16) / 41.7±6.5 (16)
LVapex_endo / 5.16±0.41 (11) a / 2.43±0.37 (11) / 50.0±7.1 (11) / 47.9±6.2 (11)
LV (all) / 4.42±0.19 (62) / 2.19±0.15 (62) / 45.4±2.7 (62) / 41.6±2.6 (62)
%dL: active cell shortening during auxotonic contraction in % of slack length; F/dtMAX and dF/dtMIN: maximum rates of force development and relaxation, respectively. Data shown as mean S.E.M., number of measurements given in round brackets; two-tailed unpaired t-test, P<0.05:
a - significantly different from LVbase_epi
b - significantly different from LVbase_endo
c - significantly different from LVapex_epi
d - significantly different from LV(all)
Table A2: Overview of regional differences in contraction dynamics measured at slack length
Region / TTP [ms] / T30 [ms]RVbase / 109.5±6.3 (13) a / 55.3±2.4 (13)
RVapex / 94.6±2.1 (14) b c d / 52.5±1.6 (14) c
RV (all) / 101.8±3.5 (27) / 53.8±1.4 (27)
LVbase_epi / 105.3±7.0 (15) / 56.2±4.0 (15)
LVbase_endo / 108.9±3.5 (17) / 51.1±1.9 (17)
LVapex_epi / 114.0±4.1 (16) / 59.9±2.4 (16) b
LVapex_endo / 113.2±5.2 (11) a / 52.0±2.2 (11) c
LV (all) / 94.6±2.1 (59) / 54.9±1.4 (59)
TTP: time-to-peak contraction; T30: time to relaxation to 30% measured from time point of peak-force. Data shown asmean S.E.M., number of measurements given in round brackets; two-tailed unpaired t-test, P<0.05:
a - significantly different from RVapex
b - significantly different from LVbase_endo
c - significantly different from LVapex_epi
d - significantly different from LVapex_endo
Table A3: Overview of the regional differences in sarcomere properties measured at cell slack length.
Region / SL0[m] / dSL
[m] / %dSL
[% of EDSL0]
RVbase / 1.89±0.01 (20) b / 0.14±0.02 (13) / 7.6±0
8 (13)
RVapex / 1.89±0.01 (15) b / 0.12±0.01 (11) / 6.4±0.7 (11)
RV (all) / 1.89±0.01 (35) / 0.13±0.01 (24) / 7.1±0.6 (24)
LVbase_e
i / 1.88±0.01 (20) b / 0.11±0.01 (11) / 5.9±0.7 (11)
LVbase_endo / 1.88±0.01 (26) b / 0.13±0.01 (11) / 7.1±0.4 (11)
LVapex_epi / 1.89±0.01 (18) b / 0.12±0.01 (14) / 6.4±0.6 (14)
LVapex_endo / 1.86±0.01 (19) / 0.15±0.01 (12) a / 8.0±0.6 (12) a
LV (all) / 1.88±0.01 (83) / 0.13±0.01 (48) / 6.9±0.3 (48)
SL0: sarcomere length at rest; dSL: absolute change in SL during contraction; %dSL: percentage change in SL compared to SL0.Data shown as mean S.E.M., number of measurements given in round brackets; two-tailed unpaired t-test, P<0.05:
a - significantly different from LVbase_epi
b - significantly different from LVapex_endo
Table A4: Overview of regional differences in sarcomere (S)dynamics measured at cell slack length.
Region / SVMAX_C[(m/ms) x 103] / SVMAX_R
[(m/ms) x 103] / STTP
[ms] / ST30
[ms]
RVbase / 3.2±0.4 (13) / 2.9±0.4 (13) / 115.9±6.2 (13) / 57.2±4.0 (13)
RVapex / 2.6±0.3 (11) / 2.7±0.4 (11) / 103.5±3.6 (11) a b / 49.5±2.1(11) b
RV (all) / 2.9±0.2 (24) / 2.8±0.3(24) / 110.3±3.9(24) / 53.7±2.5(24)
LVbase_epi / 2.5±0.3 (11) / 2.3±0.3 (11) c / 101.1±8.7 (11) b / 53.1±5.8 (11)
LVbase_endo / 2.7±0.2 (11) / 2.8±0.2 (11) / 119.6±3.6 (9) / 52.6±1.6 (11)
LVapex_epi / 2.4±0.2 (14) / 2.3±0.2 (14) c / 122.1±5.3 (14) / 57.1±2.8 (14)
LVapex_endo / 3.1±0.2 (12) b / 3.4±0.3 (12) / 117.8±6.2 (12) / 47.9±2.0 (12) b
LV (all) / 2.7±0.1 (48) / 2.7±0.1 (48) / 115.4±3.3(48) / 52.8±1.7(48)
SVMAX_C: maximal velocity of contraction; SVMAX_R: maximal velocity of relaxation;STTP: time to peak contraction (measured from the electrical trigger); ST30: time of relaxation measured from time point of peak shortening to 30% of the peak shortening; data based on the SL;data shown asmean S.E.M., number of measurements given in round brackets; two-tailed unpaired t-test, P<0.05:
a - significantly different from LVbase_endo
b - significantly different from LVapex_epi
c - significantly different from LVapex_endo
We measured dynamic cell behaviour at different preloads. Maximal rates of force development and relaxation (dF/dtMAX and dF/dtMIN) were obtained as the extremes of the first derivative of force generation. Time-to-peak (TTP, in ms) is the interval from field stimulus application to peak force development; T30is the time difference between TTP and time from stimulus to cell relaxation to 30% of peak force. Due to the variability in cell cross sectional area and distance between CF attachment positions, data were normalised through dividing length-related data by L0 or SL0, and force-related data by Sc, where applicable.All parameters are expressed as a function of normalised end-diastolic length. We studied the regional differences in slopes of a relation between the given parameter (expressed as % of the same parameter at cell slack length) and the preload. The summary of that data is presented in Table A5.
Table A5: Overview of regional differences in pre-load dependent slopes (linear approximation) of contractile behaviour (any parameter expressed as function of EDL/EDL0.
Region / dF/dtMAX (x103) slope / dF/dtMIN (x103) slope / TTP slope / T30slopeRVbase / 4.04±0.76 (13) / 4.31±0.80 (13) / 0.91±0.08 (13) c / 0.22±0.19 (13)
RVapex / 4.49±0.53 (14) a b / 4.58±0.63 (14) / 0.82±0.14 (14) a / 0.70±0.32 (14) a
RV (all) / 4.27±0.45 (27) d / 4.45±0.49 (27) / 0.86±0.08 (27) / 0.47±0.19 (27)
LVbase_epi / 2.84±0.28 (16) / 3.05±0.28 (16) / 0.94±0.06 (15) / 0.16±0.25 (15)
LVbase_endo / 3.14±0.45 (19) / 3.77±0.50 (19) / 0.90±0.12 (17) c / 0.37±0.20 (17) b
LVapex_epi / 3.49±0.48 (16) / 3.88±0.59 (16) / 1.00±0.10 (16) c / 0.01±0.22 (16) c
LVapex_endo / 3.13±0.38 (11) / 3.58±0.53 (11) / 0.70±0.15 (11) / 0.40±0.24 (11)
LV (all) / 3.16±0.21 (62) / 3.58±0.24 (62) / 0.90±0.05 (62) / 0.23±0.11(62)
dF/dtMAX and dF/dtMIN: maximum rates of force development / relaxation, respectively; TTP: time-to-peak contraction; T30: time to relaxation from time point of peak-force to 30% of the force. Data shown as mean S.E.M., number of measurements given in round brackets; ANCOVA and post-hoc two-tailed unpaired t-test, P<0.05.
a - significantly different from RVbase
b - significantly different from LVbase_epi
c - significantly different from LVapex_endo
d - significantly different from LV(all)
6. References
1. Iribe G, Helmes M, Kohl P (2007) Force-length relations in isolated intact cardiomyocytes subjected to dynamic changes in mechanical load. Am J Physiol Heart Circ Physiol 292 (3):H1487-1497
2. Nishimura S, Yasuda S, Katoh M, Yamada KP, Yamashita H, Saeki Y, Sunagawa K, Nagai R, Hisada T, Sugiura S (2004) Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions. Am J Physiol Heart Circ Physiol 287 (1):H196-202
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