Altered Myofilament Structure and Function in Dogs with Duchenne Muscular Dystrophy Cardiomyopathy
Authors: Mou Younss Ait , Lacampagne Alain , Irving Thomas C , Blot Stéphane , Ghaleh-Marzban Bijan , Magdi Yacoub , Detombe Pieter , Cazorla Olivier 
Published: July 2019

Aims

Duchenne Muscular Dystrophy (DMD) is a disease that mainly affects young mans. It is characterized by striated muscle disorder. DMD is associated with depressed heart pump function resulting from a down regulation of the left ventricular (LV) contractility. However, its effects on myofilament structure and function are poorly understood. A healthy heart is characterized by gradient of contractility spanning throughout its left ventricular (LV) wall thickness. The inner layer of the wall is known as the sub-endocardium layer (ENDO) and the outer layer is known as the sub-epicardium (EPI). The gradient of contractility is established due to the significantly high contractility of the ENDO compared to EPI. During heart failure, the contractile heterogeneity between ENDO and EPI is suppressed eliminating, thus, the gradient of contractility. Interestingly, this effect of pathology is mainly due to ENDO contractile dysfunction. These observations have maid ENDO layer to be an ideal target for heart failure treatment. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD observed in human. In the present study, we employed this animal model to evaluate the impact of DMD on the myofilament structure-function relationship. Accordingly, we isolated cardiac multicellular and single cells samples on which we evaluated myofilament lattice spacing, myofilament calcium sensitivity and contraction kinetics. It is to note that the force developed by cardiac single cell and multicellular preparations depends on their stretch status. For instance, at long length tension developed by an isolated single cardiac cell is higher than when it is maintained at slack length. During heart failure, this stretch effect on the contractility is significantly reduced particularly in the ENDO layer. Sarcomere length (SL) is the unit commonly used by investigators to evaluate muscle length. Accordingly, in the present study, we conducted our experiment on samples isolated from EPI and ENDO layers of GRMD animals at both short and long SL.

Methods and results

To evaluate the effect of muscular dystrophy on the myofilament structure, we have employed synchrotron x-rays diffraction approach to measure myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. Moreover, to evaluate transmural contractile heterogeneity in normal dog heart, myocardium samples were isolated from both EPI and ENDO left ventricular layers. Here we tested whether the lattice spacing responds differently to stretch in ENDO and EPI tissues. We obtained clear X-ray patterns from our tissue sample showing sharp equatorial reflections. As expected, spacing between thin and thick filament was reduced upon stretching. We found that d1,0 decreased linearly with SL over the SL range from 2.1 to 2.5 μm in ENDO and EPI, and that the slope of the SL–d1,0 relationship was similar in both myocardial tissue layers (Fig. 1). Despite the lack of regional lattice spacing heterogeneity, we investigated sarcomere function in isolated myocytes from the same hearts to measure myofilament calcium sensitivity. Force-pCa relationship was fitted with a modified Hill equation and EC50 ([Ca2+] generating 50% of maximal active force) was estimated. Both ENDO and EPI cardiomyocytes showed a decreased EC50 at long SL indicating increased calcium sensitivity (Fig. 2, A). To estimate myofilament length dependent activation (LDA), we computed the difference between [Ca2+] generating half of the maximal force at short (1.9 mm) and long (2.3 mm) sarcomere length (DEC50). This parameter is commonly employed to evaluate LDA. We found LDA to be higher in ENDO cardiomyocytes (Fig. 2, B) as indexed by a significantly higher DEC50 in this region. These results indicate that in dog myocardium, the higher length sensitivity of activation in the inner layer of the ventricle cannot be explained by differential interfilament lattice spacing. We evaluated myofilament Ca2+ sensitivity and LDA on permeabilized cardiomyocytes isolated from both CTRL and GRMD dogs. Myofilament calcium sensitivity was higher at short SL in ENDO GRMD myocytes compared with control dogs as indexed by a significant lower EC50 (Fig. 4, A). Differences of myofilament calcium sensitivity between CTRL and GRMD ENDO myocytes disappeared following stretch. As a result, LDA indexed by the DEC50 was lower in GRMD myocytes (Fig. 4, B). We did not find any significant difference in EPI cardiomyocytes contractile parameters. To determine whether the changes in myofilament calcium sensitivity in myocytes from failing hearts were associated with myofilament structure alteration, we performed X-ray diffraction experiments on permeabilized normal and GRMD dog ENDO myocardium at short (∼1.9 mm) and long (∼2.3 mm) sarcomere length (Fig. 4). Next, we used small -angle X-ray diffraction to assess changes in myofilament lattice structure with increasing sarcomere length in GRMD and normal dog myocardium to see if structural changes correlate with LDA. Figure 4 Panel A shows a typical CCD image of X-ray diffraction pattern. Pixel intensity was plotted and the 1,0 equatorial reflection estimated (Fig. 3, B). As observed with the first experiments (Fig. 1), the lattice spacing is reduced with stretch on CTRL myocardium (Fig. 3, C). Interestingly, the interfilament spacing was significantly higher on GRMD ENDO myocardium compared with the normal myocardium. Stretch reduced the interfilament spacing of ENDO GRMD myocardium that matches the spacing obtained on CTRL dog myocardium. The myopathy induced a myofilament lattice expansion that exceeded the physiological range. In order to test that the expansion in interfilament spacing was not due to the lack of dystrophin we also analyzed the myocardium dissected from the sub-epicardium. The higher lattice spacing in ENDO GRMD myocardium may impact myofilament calcium sensitivity and/or cross-bridge cycling kinetics. At the permeabilized single cardiomyocytes level, kinetic of tension redevelopment (ktr) was measured by mechanically disrupting force-generating cross-bridges at either sub-maximal activating solution ([Ca2+] = 1.3 μM) or at maximal calcium activation ([Ca2+] = 32 μM). Cross-bridge disruption was induced by rapid release/restretch protocol. Cardiomyocytes were perfused with activating solution, when developed force reached steady state, a rapid (2 ms) release/restretch of 20% original cell length was applied. The cell was shortly kept (20 ms) at the unload shortening prior to 100% restretch. Following the release step, force dropped to zero indicating a complete cross-bridge detachment. The restretch step, in the other hand, was characterized by an apparent monoexponential raise of force up to initial maximal force with rate constant ktr. This experimental protocol has been applied on both CTRL and GRMD permeabilized ENDO cardiomyocytes (Fig. 5). Ktr is usually used to estimate the rate of transition from weakly bound (non-force-generating) to strongly bound (force-generating) cross-bridges. Therefore, an estimation of cross-bridge cycling performance can be obtained. In ENDO cardiomyocytes from healthy dogs, ktr obtained at sub-maximal calcium activation tended to increase after stretch but it did not reach significance (Fig. 5, B). Interestingly, ktr was significantly accelerated in ENDO GRMD myocytes only at short SL (Fig. 5, A and B). Collectively, our results suggest that in myocytes from GRMD dog with heart failure there are structural changes that affect the myofilament contractile properties.

Conclusions

At short SL myopathy induces an excessive expansion of the myofilament lattice spacing that may affect myosin heads orientation. This myofilament restructuration improves weak to strong cross-bridge transition as indicated by ktr acceleration. The positive cooperative activation of thin filament through strongly bound cross-bridges improves myofilament Ca2+ sensitivity and overall cardiomyocyte active tension. Additional experiment need to be conducted to better understand the interconnection between myofilament lattice spacing and performance in myopathy induced heart failure of large animal model.