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. 2017 Dec 22:8:1103.
doi: 10.3389/fphys.2017.01103. eCollection 2017.

Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy

Ilse A E Bollen  1 , Marijke van der Meulen  2 , Kyra de Goede  1 , Diederik W D Kuster  1 , Michiel Dalinghaus  2 , Jolanda van der Velden  1   3
Affiliations

Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy

Ilse A E Bollen et al. Front Physiol. .

Abstract

Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM) is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

Keywords: cardiomyopathy; dilated; heart failure; hypocontractility; myofibril density; pediatrics; titin.

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Figures

Figure 1
Figure 1
Baseline characteristics. (A) Maximal force was significantly lower in pediatric CM (23.8 ± 1.1, N = 11, n = 78) compared to controls (37.4 ± 2.6, N = 6, n = 27, p < 0.0001). (B) Fpass was significantly lower in pediatric CM (N = 11, n = 40) compared to controls (N = 10, n = 29, p < 0.0001) over a range of sarcomere lengths. (C) A leftward shift of the relative force vs. [Ca2+] indicates higher myofilament Ca2+-sensitivity in pediatric CM (N = 11, n = 38) compared to controls (N = 5, n = 12, p < 0.0001). (D) The absolute force development over a range of [Ca2+] showed pediatric CM have impaired maximal force development at saturating [Ca2+], increased force development at lower [Ca2+] and a decreased Fpass. (E) Ca2+-sensitivity was significantly higher at sarcomere lengths of 1.8 and 2.2 μm in pediatric CM (N = 11, n = 38) compared to controls (N = 5, n = 12, p < 0.0001). (F) Length-dependent activation, measured as ΔEC50 was significantly lower in pediatric CM (0.60 ± 0.05, N = 11, n = 38) compared to controls (0.86 ± 0.09, N = 5, n = 12, p = 0.007). N, number of samples; n, number of cardiomyocytes measured. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray (A,F). **p < 0.01, ****p < 0.0001 vs. controls.
Figure 2
Figure 2
Titin isoform composition has limited effect on contractility in pediatric CM. (A) Separation of titin N2BA and N2B with gel electrophoresis. (B) N2BA/N2B ratio was not significantly different between pediatric CM (0.62 ± 0.12, N = 11) and controls (0.53 ± 0.03, N = 15). (C) Length-dependent activation was mostly impaired in pediatric CM patients who had higher N2BA/N2B ratio (N2BA/N2B > 0.65, N = 5, n = 19) compared to pediatric CM patients who had lower N2BA/N2B ratio (N2BA/N2B < 0.4, N = 6, n = 19). Dotted line indicates control values. (D) Mean ΔEC50 per sample plotted against the N2BA/N2B ratio did not show a significant correlation between ΔEC50 and N2BA/N2B. (E) There was no difference in Fpass between pediatric CM patients who had higher N2BA/N2B ratio (N2BA/N2B > 0.65, N = 5, n = 18) and pediatric CM patients who had lower N2BA/N2B ratio (N2BA/N2B < 0.4, N = 6, n = 22). (F) N2BA/N2B ratio was not significantly related to age. (G) No significant correlation was found between N2BA/N2B and LVEDD z-score. (H) No significant correlation was found between N2BA/N2B and LVESD z-score. (I) No significant correlation was found between N2BA/N2B and LVPWd z-score. (J) A significant correlation was found between N2BA/N2B ratio and LVPWs z-score (p < 0.05). N, number of samples; n, number of cardiomyocytes measured. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray (B–D).
Figure 3
Figure 3
Hypophosphoryalation in pediatric CM compared to controls. (A) ProQ staining identifying phosphorylated proteins of pediatric CM and control samples. (B) Corresponding SYPRO staining identifying proteins of pediatric CM and control samples. (C) cTnI phosphorylation was significantly lower in pediatric CM (N = 11) compared to controls (N = 13, p < 0.0001). (D) Phostag showed separation of non-, mono-, and bisphosphorylated cTnI. (E) While controls (N = 11) showed predominantly mono- and bisphosphorylated cTnI, pediatric CM samples (N = 11) showed mostly non-phosphorylated cTnI. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray. ****p < 0.0001 vs. controls.
Figure 4
Figure 4
Restoration of sarcomere function after incubation with exogenous PKA. (A) Exogenous PKA restored myofilament Ca2+-sensitivity in pediatric CM (N = 11, n = 30) to control (n = 5, n = 12) values. (B) Exogenous PKA eliminated the difference in length-dependent activation (ΔEC50) between pediatric CM patients with higher N2BA/N2B ratio (N2BA/N2B > 0.65, N = 5, n = 14) and pediatric CM patients who had lower N2BA/N2B ratio (N2BA/N2B < 0.4, N = 6, n = 16) (shown relative to control value). Dotted line indicates control values. (C) There was no difference between patients with high or low N2BA/N2B ratio with respect to ΔEC50 after incubation with exogenous PKA. (D) Mean ΔEC50 per sample measured after incubation with exogenous PKA plotted against the N2BA/N2B ratio did not show a significant correlation between ΔEC50 and N2BA/N2B. (E) Exogenous PKA did not affect Fpass in controls or pediatric CM. Fpass remained significantly lower in pediatric CM compared to controls. (F) There was no difference in Fpass after incubation with exogenous PKA between pediatric CM patients who had higher N2BA/N2B ratio (N2BA/N2B > 0.65, N = 5, n = 13) and pediatric CM patients who had lowerN2BA/N2B ratio (N2BA/N2B < 0.4, N = 6, n = 18). N, number of samples; n, number of cardiomyocytes measured. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray (B–D).
Figure 5
Figure 5
Myofibril density is decreased in pediatric CM. (A) Electron microscopy images of 2 control samples and 2 pediatric CM samples. (B) Myofibril density was significantly lower (P < 0.0001) in pediatric CM (N = 10, 45.3 ± 1.4%) compared to controls (N = 10, 57.3 ± 1.8%). (C) Fmax normalized for myofibril density of corresponding sample did not differ significantly between pediatric CM (N = 10, n = 71) and controls (N = 6, n = 27). (D) Fpass normalized for myofibril density of corresponding sample was not significantly different between pediatric CM (N = 10, n = 37) and controls (N = 9, n = 25). N, number of samples; n, number of cardiomyocytes measured. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray. ****p < 0.0001 vs. controls.
Figure 6
Figure 6
Protein quality control system in pediatric CM. (A) Representative blot images for HSP27 expression. (B) Representative blot images for HSP70 expression. (C) Expression of HSP27 was not altered in pediatric CM (N = 11) compared to controls (N = 11). (D) Expression of HSP70 was not altered in pediatric CM (N = 11) compared to controls (N = 11). (E) Representative blot images for LC3BI and LC3B-II expression. (G) Expression of LC3B-I/LC3-BII was significantly (p < 0.05) reduced in pediatric CM (N = 11) compared to controls (N = 11). (F) Representative blot images for p62 expression. (H) Expression of p62 was not altered in pediatric CM (N = 11) compared to controls (N = 7). GAPDH was used a loading control in the HSP27, HSP70, and p62 blots. Measurements obtained from samples derived from patients with non-compaction cardiomyopathy are indicated in gray. *p < 0.05, vs. controls.
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