Effect of dantrolene on calcium transients.

<p>A) Depolarization with 60 mM KCl and 20 mM caffeine in normal buffer, followed by exposure to sequential exposures to caffeine in the presence of 10 µM dantrolene. B) The amplitude of the caffeine transient in the absence or presence of dantrolene. Note that dantrolene almost completely inhibits the ability of the myotubes to respond to caffeine (p<0.05) and lowers the cytoplasmic calcium concentration of resting myotubes. C) Indo-1 fluorescence at increasing calcium concentration, in the presence or absence of 10 µM dantrolene. Note that there was no direct effect of dantrolene on Indo-1 fluorescence.</p

Depolarisation with KCl.

<p>Representative experiment where skin-derived equine myotubes were subjected repeatedly to depolarization with 60 mM KCl (red line). 10 mM caffeine responses (blue line) are shown for comparison. Note that responses are reproducible and near maximal within approximately 4 minutes.</p

Caffeine dose response.

<p>Histogram of Indo-1 response to increasing concentrations of caffeine in adenovirally-transduced equine myotubes. Results show mean (+/−1 SEM) derived from 40 myotubes from 2 control horses sequentially exposed to 5, 10 and 20 mM caffeine at 2 and 3 weeks’ differentiation.</p

Effects of Cl, Met and MetCl therapy on cardiomyocyte contractility (A–C) and Ca²⁺ handling (D–F) from non-transplanted failing hearts, measured using Indo-1 and Ionoptix system are shown.

<p>Full recovery of speed of sarcomeric contraction (B), relaxation (C), Ca²⁺ transient amplitude (D) and speed of Ca²⁺ release (E) caused by Met therapy, either alone or in combination with Cl, and lack of improvement following Cl mono-therapy is shown. Superiority of Met mono-therapy in recovering depressed SR Ca²⁺ content is also shown (F). *P<0.05, **P<0.01 and ***P<0.001.</p

Full recovery of HF-induced depression of L-type Ca²⁺ current (A) and t-tubule density (B) caused by Cl, and lack of improvement in these parameters following Met and MetCl therapy are shown.

<p>***P<0.001. Representative di-8-Anepps stained cells from sham (C), HF (D), HF+Cl (E), HF+Met (F) and HF+MetCl (G) groups. Data for the Met group has been previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092909#pone.0092909-Navaratnarajah1" target="_blank">[22]</a> and added here for comparison.</p

Effect of Cl, Met and combined MetCl treatment on heart weight (HW) (A) and cardiomyocyte volume measured using confocal microscopy (B) during unloading (MUHF).

<p>Prevention of MU-induced cardiac and cardiomyocyte atrophy is achieved by Met and not Cl, with combined MetCl therapy increasing atrophy. *P<0.05, **P<0.01 and ***P<0.001, (HW and cardiomyocyte volume data acquired from 8 and 4 hearts per group, respectively). Effect of Cl, Met and combined MetCl therapy on heart weight∶body weight ratio (HW∶BW) (C) and cardiomyocyte volume (D). HF-induced cardiac hypertrophy was enhanced by Cl therapy but this effect disappeared during combined MetCl therapy. HF-induced myocyte hypertrophy was partially attenuated by Met, but this effect was lost during combination MetCl therapy (HW and cardiomyocyte volume data acquired from 8 and 4 hearts per group, respectively).</p

MU-induced recovery of depressed L-type Ca²⁺ current (A) and t-tubule density (B).

<p>Maintenance of L-type Ca²⁺ current recovery by combined MetCl therapy, and antagonism of such recovery by Cl or Met mono-therapy (A) is shown, along with antagonism of t-tubule density recovery by all treatments (B). *P<0.05, **P<0.01 and ***P<0.001. Representative di-8-Anepps stained cells from sham (C), HF (D), MUHF (E), MUHF+Cl (F), MUHF+Met (G) and MUHF+MetCl (H) groups are shown. Data for the Met group has been previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092909#pone.0092909-Navaratnarajah1" target="_blank">[22]</a> and added here for comparison.</p

Effect of Cl, Met and combined MetCl therapy on contractile function in non-transplanted failing hearts, EF (A) and FS (B): No difference in baseline values in treatment groups was seen (light grey bars).

<p>Average values at end of treatment period are shown (black bars): Cl-treated group showed improved EF and FS compared to untreated HF group, and improvement in EF was further enhanced in MetCl group. Met-induced improvement in EF and FS was not statistically significant. ***P<0.001 (n = 8 per group).</p

Anatomical and cell volume data (16 weeks after LCA ligation).

<p>**P<0.01,</p><p>***P<0.001 vs. sham.</p><p>Anatomical and cell volume data (16 weeks after LCA ligation).</p

Effects of Cl, Met and MetCl therapy on cardiomyocyte contractility (A–C) and Ca²⁺ handling (D–F) during mechanical unloading (MUHF), measured using Indo-1 and Ionoptix system.

<p>Cl's enhancement of MU-induced recovery of speed of sarcomeric contraction (B) and Met's antagonism of MU-induced improvement in speed of relaxation (C) are shown. Cl and Met's enhancement of MU-induced recovery of Ca²⁺ transient amplitude (D), speed of Ca²⁺ release (E) and SR Ca²⁺ content (F), and lack of enhancement during combined MetCl therapy is shown. *P<0.05, **P<0.01 and ***P<0.001. Representative traces of sarcomeric contractions (G) and Ca²⁺ transients (H) are shown.</p