Incomplete recovery of myocyte contractile function despite improvement of myocardial architecture with left ventricular assist device support

Abstract

Background: Unloading a failing heart with a left ventricular assist device (LVAD) can improve ejection fraction (EF) and LV size; however, recovery with LVAD explantation is rare. We hypothesized that evaluation of myocyte contractility and biochemistry at the sarcomere level before and after LVAD may explain organ-level changes.

Methods and results: Paired LV tissue samples were frozen from 8 patients with nonischemic cardiomyopathy at LVAD implantation (before LVAD) and before cardiac transplantation (after LVAD). These were compared with 8 nonfailing hearts. Isolated skinned myocytes were purified and attached to a force transducer, and dimensions, maximum calcium-saturated force, calcium sensitivity, and myofilament cooperativity were assessed. Relative isoform abundance and phosphorylation levels of sarcomeric contractile proteins were measured. With LVAD support, the unloaded EF improved (10.0±1.0% to 25.6±11.0%, P=0.007), LV size decreased (LV internal dimension at end diastole, 7.6±1.2 to 4.9±1.4 cm; P<0.001), and myocyte dimensions decreased (cross-sectional area, 1247±346 to 638±254 μm(2); P=0.001). Maximum calcium-saturated force improved after LVAD (3.6±0.9 to 7.3±1.8 mN/mm(2), P<0.001) implantation but was still lower than in nonfailing hearts (7.3±1.8 versus 17.6±1.8 mN/mm(2), P<0.001). An increase in troponin I (TnI) phosphorylation after LVAD implantation was noted, but protein kinase C phosphorylation of TnI decreased. Biochemical changes of other sarcomeric proteins were not observed after LVAD.

Conclusions: There is significant improvement in LV and myocyte size with LVAD, but there is only partial recovery of EF and myocyte contractility. LVAD support was associated only with biochemical changes in TnI, suggesting that alternate mechanisms might contribute to contractile changes after LVAD and that additional interventions may be needed to alter biochemical remodeling of the sarcomere to further enhance myofilament and organ-level recovery.

Figure 1

Photomicrograph example of an isolated skinned myocyte fragment attached to the force transducer and motor in relaxing solution for mechanical contractile experiments. The visible sarcomere striations were set at 2.1μm for all myocyte experiments.

Figure 2

(a) Representative tracing of force measurements from the experimental protocol. After the myocyte had achieved a steady force in the calcium containing activation solution, it was rapidly step shortened to break cross-bridges (top tracing) and the maximum force for the particular activation solution was then recorded (bottom tracing). (b) Representative plot of the force-calcium relation. Force is shown plotted against the inverse log of the calcium concentration (pCa). The Fmax (the maximal calcium saturated developed force normalized to the cross-sectional area of the myocyte), pCa50 (the calcium concentration at which the force is half maximal, a measure of calcium sensitivity), and Hill coefficient (the slope of the calcium-force relation, an index of myofilament cooperative activation) for each myocyte were derived from these force-pCa curves.

Figure 2

(a) Representative tracing of force measurements from the experimental protocol. After the myocyte had achieved a steady force in the calcium containing activation solution, it was rapidly step shortened to break cross-bridges (top tracing) and the maximum force for the particular activation solution was then recorded (bottom tracing). (b) Representative plot of the force-calcium relation. Force is shown plotted against the inverse log of the calcium concentration (pCa). The Fmax (the maximal calcium saturated developed force normalized to the cross-sectional area of the myocyte), pCa50 (the calcium concentration at which the force is half maximal, a measure of calcium sensitivity), and Hill coefficient (the slope of the calcium-force relation, an index of myofilament cooperative activation) for each myocyte were derived from these force-pCa curves.

Figure 3

12% 1D-SDS-PAGE images stained with a total phosphoprotein stain (ProQ Diamond Phosphoprotein Gel Stain) in panel A and a total protein stain (BioSafe Coomassie Blue) in panel B demonstrate the increase in total TnI phosphorylation with LVAD support and higher MyBPC phosphorylation in both failing samples compared with nonfailing donors. Sarcomeric protein phosphorylation levels were calculated by dividing the optical density of the each sarcomeric protein on the ProQ diamond gel by the optical density of the same protein on the Coomassie gel. MyBPC was normalized to MLC-1 as noted in the text. NF=nonfailing, MyBPC=myosin binding protein C, TnT=troponin T, Tm=tropomyosin, TnI=troponin I, MLC-1=myosin light chain 1, MLC-2=myosin light chain 2

Figure 4

Western blots of phosphorylated troponin I (TnI) demonstrate while there were no significant differences in Serine 22,23 site specific phosphorylation of TnI, Serine 43 site specific phosphorylation of TnI decreased after LVAD. Samples were separated on 12.5% SDS-PAGE and probed with antibodies against phosphoserine 22/23, phosphoserine 43, and cardiac TnI (for total protein). NF=nonfailing.

Figure 5

6% 1D-SDS-PAGE separation of α and β myosin before and after LVAD demonstrates that only the β-isoform is expressed in both groups. The last lane is a mixture of 50% nonfailing human with 50% nonfailing mouse and illustrates adequate separation of α and β myosin isoforms.