This study shows that chronic, intermittent P188 may be effective in protecting cardiac function in a subcutaneous low dose isoproterenol stress model over a 4 week period in dystrophin deficient hearts. Mdx mice treated with P188 during exposure to isoproterenol were able to maintain increased systolic function (46 ± 6 vs. 35 ± 6 SF%; p < 0.01) compared to untreated isoproterenol exposed mdx mice suggesting that chronic, intermittent injections of intraperitoneal P188 can stabilize cardiac muscle cell membranes in vivo. These results also provide a model for the development of mild cardiomyopathy and cardiac fibrosis in 2 month old mdx mice over a period of 4 weeks and allows for quicker screening of potential drugs targeting cardiac function and fibrosis.
We also showed that mdx mice remain exquisitely sensitive to in vivo cardiac stress from the beta agonist administration. All mdx mice died within 24 hours at doses of 30, 15, 10, 5 and 1 mg/kg/day and could only tolerate a six-fold decreased isoproterenol dose (0.5 mg/kg/day) compared to BL10 control mice. Even at this dosing, mdx mice had a shortening fraction of 42 ± 6% at 24 hours that decreased to 38 ± 5% at 2 weeks and 35 ± 6% at 4 weeks. P188 treatment did not improve survival at the higher levels of isoproterenol. This data suggests that the effectiveness to chronic intraperitoneal injections of P188 is limited and cannot stabilize cardiac cells as well as acute, intravenous administration.
P188 was previously studied in dystrophin deficient animal models and shown to prevent acute cardiac failure after dobutamine-induced cardiac stress in mdx mice. These studies utilized acute, intravenous administration . Using chronic, intermittent dosing, our non-invasive in vivo cardiac stress model of subcutaneous isoproterenol administration and cardiac assessment using echocardiography demonstrated similar results. Yasuda et al. (2005) concluded that the primary effect of P188 was to acutely restore myocyte compliance and improve end-diastolic volumes. While we did not measure these parameters, increased systolic function that was equal to BL10 mice demonstrated an increased capacity to tolerate cardiac stress and is likely related to improved myocyte compliance. P188 can also decrease blood viscosity by stabilizing red blood cell membranes; decreasing red blood cell adhesion; and improving microvascular blood flow [19, 20]. These changes can decrease effective afterload and are potential physiological mechanisms that could improve heart rates and cardiac systolic function in P188 treated mice.
Townsend et al. (2010) recently reported that a chronic 8 week infusion of P188 (60 mg/kg/h) in golden retriever muscular dystrophy (GRMD) dogs showed significantly decreased cardiac fibrosis and prevented ventricular dilation . Our study did not show a significant decrease in cardiac fibrosis in P188 treated mice (3.0% vs. 2.9%). This may be related to our single daily dosing protocol that provided sufficient P188 to partially stabilize some cardiomyocytes and improve cardiac function, but not enough to prevent significant myocyte loss and fibrosis. The GRMD model also shows a more severe, naturally occurring cardiomyopathy which is difficult to replicate in the mouse model, even with isoproterenol exposure. Thus, fibrotic signaling pathways in the GRMD model were increased compared to the mdx mouse, and P188 therapy showed more significant modulation. The mdx mouse was so sensitive to isoproterenol stress that we were unable to test higher doses that could have increased fibrosis, without mortality, to better evaluate the anti-fibrotic effectiveness of P188.
Our study also did not show ventricular dilation, but rather displayed decreased left ventricular internal dimensions. This was secondary to increased left ventricular wall thickness and mass, seen in both P188 treated and untreated mice from the increased cardiac workload. Previous studies have shown both dilated and non-dilated cardiomyopathy in mdx mice [2, 3]. In the presence of isoproterenol, untreated mdx mice were able to show cardiac hypertrophy similar to BL10 mice and this is likely related to compensatory mechanisms that are still present in the 3 month old mice that become decreased with age.
We unexpectedly demonstrated a significant increase in Evan's blue dye (EBD) positive fibers in P188 treated mdx hearts. Quilan et al. (2006) also found similar unexpected results studying skeletal muscles in exercised mdx mice, showing equal or increased %-EBD penetration in P188 treated rectus femoris muscle . However, although we found an increase in EBD positive fibers, these hearts showed increased systolic function compared to untreated hearts. Based on these findings, we propose that P188 can stabilize mildly damaged cells, especially at lower effective doses. These cells remain functional but become EBD positive on histology. Also, there is the potential that P188 facilitates EBD entry into cells without compromising function.
We showed significantly lower blood pressures in mdx mice at baseline compared to controls. This was previously reported in 9 month old mdx mice . P188 treatment had no effect on blood pressure levels, as was also seen in the study by Townsend et al. (2010) . The mechanism for decreased blood pressures is not clear at this time and may be related to cardiac or vascular dysfunction, or both . Previous studies demonstrated that the myocardium can generate more force after treatment with P188 [9, 21]. This study also demonstrates improved cardiac function after P188 treatment. However, we do not see a corresponding effect in blood pressure. Thus, there is likely still an undetermined vascular component to decreased systolic blood pressures in mice that was not compensated for at this level of improved cardiac function. This supports previous literature that shows that mdx mice have abnormal vascular endothelium responses and blood pressure regulation [24, 25].
This study did not show an improvement in skeletal muscle specific force using in vitro measurements. Ng et al. (2008) found that P188 addition to the muscle bathing solution reduced the force deficit in the isolated, whole lumbrical muscle . Previous studies also showed that P188 restored compliance to isolated cardiac myocytes in solution, but these studies utilized acute administration of P188. When myocytes were used after a P188 washout period, properties returned to those of untreated animals [9, 21]. We did not measure P188 serum levels and cannot estimate peak levels of exposure. While benefits were seen in cardiac function, no significant improvements in skeletal muscle function or cardiac fibrosis were seen in this study secondary to the chronic, intermittent dosing schedule. Thus, there is mounting evidence that P188 is most effective during acute administration in both cardiac and skeletal muscle.