Ranolazine suppressed in vivo VA inducibility and severity in mouse hearts with regional IR injury
In the in vivo electrophysiological studies, we acquired data from 7, 7, 8, and 7 mice in the db/db C, db/db R, db/+ C, and db/+ R groups, respectively. The effective refractory period was significantly longer in the db/db C mice than in the db/db R, db/+ C, and db/+ R groups (74 ± 17 vs. 62 ± 18, 58 ± 18, and 60 ± 11 ms, respectively; P = 0.034). Figure 1 summarizes the result of VT inducibility and severity. VT was inducible in 7 of 7, 5 of 7, 8 of 8 and 5 of 7 mice in the db/db C, db/db R, db/+ C, and db/+ R groups, respectively (P = NS, Fig. 1A). But the percentage of VT-induced episodes was higher in the db/db C group compared to the db/+ C group by burst pacing protocol (P = 0.003). Pretreatment of ranolazine significantly reduced the percentage of VT episodes by both burst pacing and extrastimulus pacing protocols in both db/db and db/+ groups (Fig. 1B). Figure 1C shows the number and percentage of VT episodes < 10 beats, between 10 to 30 sec, and > 30 beats in each group. Even if the distribution of VT duration shows most of the VT lasted less than 10 beats, db/db C mice were significantly more vulnerable to longer VTs: Longer VT (> 30 beats) was induced in 5 of 7 (db/db C, the longest 180 beats), 1 of 7 (db/db R, the longest 50 beats), 1 of 8 (db/+ C, the longest 72 beats) and 1 of 7 (db/+ R, the longest 66 beats) hearts (P = 0.031).. A representative example of pacing-induced VT in a db/db C mouse heart is shown in Fig. 1D.
Optical Mapping Studies
Ranolazine effects on APD80 and CaiTD80
In the optical mapping studies, we acquired data from 11, 10, 11, and 10 mice in the db/db C, db/db R, db/+ C, and db/+ R groups, respectively. As shown in Fig. 2A, the db/db C group tended to have a longer APD80 than the db/+ C group. APD80 in the db/db C group was significantly longer than that in the db/db R group, but there was no significant difference between the db/+ C and db/+ R groups. In addition, APD80 in the IR zone was significantly longer than that in the non-IR zone in the ranolazine non-given groups, but not in the ranolazine given groups (Table 1). The APD80 dispersion was significantly different among the 4 groups and the db/db C group had the largest APD80 dispersion: at PCL = 200 ms: 27 ± 8, 22 ± 7, 17 ± 5, 16 ± 3 ms in db/db C, db/+ C, db/db R, db/+ R groups, respectively (P = 0.002); at PCL = 100 ms: 17 ± 5, 17 ± 3, 13 ± 3, 13 ± 4 ms in db/db C, db/+ C, db/db R, db/+ R groups, respectively (P = 0.041). Similarly, CaiTD80 in the db/db C group was significantly longer than that in the db/db R group, and CaiTD80 in the IR zone was significantly longer than that in the non-IR zone in the ranolazine non-given groups, but not in the ranolazine given groups. The difference of CaiTD80 dispersion was insignificant at PCL = 200 ms: 23 ± 7, 19 ± 7, 17 ± 8, and 16 ± 6 ms (P = 0.217); but was significant at PCL = 100 ms: 18 ± 7, 18 ± 7, 10 ± 5, and 11 ± 5 ms (P = 0.028) in the db/db C, db/+ C, db/db R, and db/+ R groups, respectively. These findings indicated that ranolazine shortened APD80 and CaiTD80 in db/db mouse hearts, reduced the differences of APD80 and CaiTD80 between non-IR and IR zones, and attenuated the APD80 and CaiTD80 heterogeneity in both db/db and db/+ mouse hearts. Figure 2B shows representative examples of APD80 and CaiTD80 maps of the four groups. The db/db C mouse heart had the longest APD80 and CaiTD80, which were longer in the IR zone than in the non-IR zone.
Table 1
Electrophysiological effects of ranolazine in isolated Langendorff-perfused mouse hearts after IR injury.
| APD80 (ms) | CaiTD80 (ms) | CV (cm/s) |
200 ms | 100 ms | 200 ms | 100 ms | 200 ms | 150 ms | 120 ms | 100 ms | 90 ms | 80 ms | 70 ms | 60 ms |
db/db C (N = 11) | Non-IR | 82 ± 9* | 52 ± 7* | 91 ± 7* | 69 ± 5* | 78 ± 13* | 76 ± 13* | 74 ± 14* | 70 ± 13* | 64 ± 12* | 57 ± 13* | 51 ± 12* | 45 ± 12* |
IR | 86 ± 9* | 56 ± 6* | 100 ± 9* | 77 ± 8* | 68 ± 18* | 61 ± 16* | 57 ± 15* | 54 ± 14* | 50 ± 12* | 47 ± 11* | 41 ± 10* | 37 ± 10* |
db/db R (N = 10) | Non-IR | 70 ± 7 | 48 ± 6 | 84 ± 11 | 65 ± 6 | 88 ± 5 | 84 ± 8 | 80 ± 9 | 75 ± 8 | 74 ± 11 | 72 ± 12 | 64 ± 12 | 62 ± 10 |
IR | 73 ± 10 | 51 ± 6 | 89 ± 9 | 67 ± 5 | 82 ± 9 | 78 ± 8 | 76 ± 7 | 70 ± 6 | 67 ± 7 | 61 ± 6 | 58 ± 5 | 53 ± 6 |
db/+ C (N = 11) | Non-IR | 75 ± 4 | 46 ± 4* | 89 ± 7* | 68 ± 3* | 83 ± 15 | 78 ± 13* | 75 ± 10* | 70 ± 12* | 67 ± 13* | 60 ± 12* | 57 ± 13* | 52 ± 11* |
IR | 78 ± 4 | 54 ± 4* | 96 ± 10* | 73 ± 6* | 69 ± 12 | 64 ± 13* | 59 ± 11* | 55 ± 9* | 51 ± 9* | 46 ± 8* | 43 ± 7* | 38 ± 7* |
db/+ R (N = 10) | Non-IR | 71 ± 10 | 46 ± 2 | 84 ± 6 | 65 ± 4 | 90 ± 25 | 89 ± 25 | 85 ± 27 | 83 ± 28 | 80 ± 25 | 75 ± 23 | 67 ± 19 | 61 ± 17 |
IR | 73 ± 14 | 49 ± 5 | 86 ± 10 | 66 ± 5 | 86 ± 22 | 82 ± 21 | 80 ± 22 | 75 ± 24 | 72 ± 21 | 68 ± 22 | 64 ± 21 | 56 ± 16 |
Values are mean ± SD. APD80, action potential duration at 80% repolarization; CaiTD80, effective refractory period; CV, conduction velocity; IR, ischemia-reperfusion. * indicates P < 0.05 for non-IR vs. IR. |
Ranolazine effects on Cai decay
Figure 3A shows the summarized results of Cai decay tau value among the four groups. Cai decay time was the longest in the db/db C group (P = 0.013). Ranolazine shortened the tau value more significantly in db/db mouse hearts (from 35.8 ± 3.8 ms to 31.5 ± 3.4 ms; P = 0.001) than in db/+ mouse hearts (from 32.8 ± 3.5 ms to 30.8 ± 3.9 ms; P = 0.083). Furthermore, Cai decay time was longer in the IR zone than in the non-IR zone. Ranolazine ameliorated the differences in the tau values between the non-IR and IR zones. As shown in Fig. 3A, the P values were increased from 0.044 to 0.055 (db/db C vs. db/db R) and from 0.066 to 0.118 (db/+ C vs. db/+ R). A representative example of Cai decay at the non-IR and IR zones in the four groups is shown in Fig. 3B.
Ranolazine effects on CV
Table 1 and Fig. 4A summarize the effects of 1-week ranolazine pretreatment on CV in mouse hearts with acute regional IR injury. Among the four groups, CVnon−IR did not differ significantly at all PCLs. However, CVIR was significantly different at PCLs of 150, 120, 100, 90, 80, 70, and 60 ms (P = 0.022, 0.007, 0.013, 0.003, 0.004, 0.002, and 0.002, respectively). Post hoc analysis showed that the difference in CVIR was mainly due to the pretreatment of ranolazine. Ranolazine ameliorated CVIR slowing to improve conduction inhomogeneity. As shown in Fig. 4A, the difference between CVIR and CVnon−IR was significant in the db/db C and db/+ C groups, but insignificant in the db/db R and db/+ R groups. Figure 4B shows an example of isochrone maps in the four groups. At PCL = 60 ms, the CVIR was slower in the db/db C (44 cm/s) and db/+ C (46 cm/s) mice than in the db/db R (58 cm/s) and db/+ R (70 cm/s) mice, and the difference between CVIR and CVnon−IR was also greater in the db/db C (17 cm/s) and db/+ C (17 cm/s) mice than in the db/db R (13 cm/s) and db/+ R (5 cm/s) mice.
Induction of spatially concordant and discordant alternans
Although spatially concordant alternans (SCA) could be provoked in all hearts in the four groups, the longest PCL required to provoke SCA was significantly shorter in the ranolazine groups (107 ± 15, 86 ± 5 ms in the db/db C, db/db R groups, respectively; 102 ± 15, 79 ± 17 ms in the db/+ C, db/+ R groups, respectively; P < 0.001). Similarly, spatially discordant alternans (SDA) could be provoked in all hearts, and the longest PCL required to provoke SDA was significantly shorter in the ranolazine groups (90 ± 12, 69 ± 11 ms in the db/db C, db/db R groups, respectively; 80 ± 14, 67 ± 9 ms in the db/+ C, db/+ R groups, respectively; P < 0.001). A representative example was shown in supplementary Fig. 1.
VA inducibility
VA inducibility was significantly different among the four groups: VA was induced in 10 of 11 (db/db C), 3 of 10 (db/db R), 7 of 11 (db/+ C) and 2 of 10 (db/+ R) hearts (P = 0.004). Figure 5 illustrates VT induction in a db/db C mouse heart. Figures 5A and 5B show images of IR creation and the mapping field, respectively. Figure 5C shows the Vm recordings at sites “a” (rotor anchoring site on a nodal line, Fig. 5E) and “b” (left ventricular base) during VT induction. Extrastimulus pacing led to dispersion of refractoriness and unidirectional conduction block (frame 310; Fig. 5D), and reentrant wavefronts were initiated after pacing (frames 346–509). During the initiation of VT, the core of reentrant wavefronts anchored at site “a,” where fragmented Vm transient is shown (Fig. 5C). Post hoc analysis revealed that ranolazine effectively suppressed the VA inducibility in both db/db and db/+ mouse hearts with acute regional IR injury (Fig. 5F)
Protein Expression
To elucidate the roles of Ca2+-handling proteins, Na+ channel, and Cx43 in the antiarrhythmic mechanisms of ranolazine, we measured and compared the levels of the associated proteins between the IR and non-IR zones. The results are presented in Fig. 6 and supplementary Fig. 2. In db/db C hearts, the expression levels of pThr17-phospholamban, calsequestrin 2, and SCN5A in the IR zone were significantly lower than those in the non-IR zone. Ranolazine pretreatment attenuated the downregulation of these proteins in the IR zone by acute IR injury. In db/+ C hearts, the expression level of pThr17-phospholamban was significantly lower than that in the non-IR zone, which was attenuated by ranolazine.
Ranolazine effects on INa,L in cardiomyocytes from db/db and db/+ mice with acute IR injury
The db/db cardiomyocytes expressed a greater INa,L density (0.420 ± 0.214 pA/pF, n = 93) than the db/+ cells (0.209 ± 0.056 pA/pF, n = 51, P < 0.001). As shown in Fig. 7A, ranolazine therapy significantly decreased the density of INa,L in both db/db mice (0.497 ± 0.219 pA/pF, n = 61 in db/db C vs. 0.272 ± 0.096 pA/pF, n = 32 in db/db R, P = 0.034) and db/+ mice (0.229 ± 0.044 pA/pF, n = 15 in db/+ C vs. 0.201 ± 0.059 pA/pF, n = 36 in db/+ R, P = 0.047), but the density of INa,L in db/db R group was still higher than those in db/+ C (P = 0.048) and db/+ R (P = 0.037) groups. There was significant difference of INa,L density between IR and non-IR cardiomyocytes in the db/db C group (0.655 ± 0.168 pA/pF, n = 23 vs 0.402 ± 0.189 pA/pF, n = 38, P < 0.001), but not in the db/db R group (0.291 ± 0.076 pA/pF, n = 20 vs 0.240 ± 0.120 pA/pF, n = 12, P = 0.077).