Author + information
- Received January 27, 2014
- Accepted April 16, 2014
- Published online July 8, 2014.
- Emmy Manders, MSc∗,†,
- Harm-Jan Bogaard, MD, PhD∗,
- M. Louis Handoko, MD, PhD†,‡,
- Marielle C. van de Veerdonk, MD∗,
- Anne Keogh, MD§,
- Nico Westerhof, PhD∗,†,
- Ger J.M. Stienen, PhD†,‖,
- Cristobal G. dos Remedios, PhD, Dsc¶,
- Marc Humbert, MD, PhD#∗∗,††,
- Peter Dorfmüller, MD, PhD#,††,‡‡,
- Elie Fadel, MD, PhD#,††,§§,
- Christophe Guignabert, PhD#,††,
- Jolanda van der Velden, PhD†,‖‖,
- Anton Vonk-Noordegraaf, MD, PhD∗,
- Frances S. de Man, PhD∗∗ ( and )
- Coen A.C. Ottenheijm, PhD†∗∗ ()
- ∗Department of Pulmonology, Vrije Universiteit (VU) University Medical Center, Amsterdam, the Netherlands
- †Department of Physiology, VU University Medical Center, Amsterdam, the Netherlands
- ‡Cardiology Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands
- §Heart Transplant Unit, St. Vincent's Hospital, Sydney, Australia
- ‖Department of Physics and Astronomy, VU University, Amsterdam, the Netherlands
- ¶Muscle Research Unit, Bosch Institute, The University of Sydney, Sydney, Australia
- #University of Paris-Sud, Faculté de Médecine, Le Kremlin-Bicêtre, France
- ∗∗Assistance Publique–Hôpitaux de Paris, Service de Pneumologie, Département Hospitalo-Universitaire, Thorax Innovation (DHU TORINO), Hôpital Bicêtre, Le Kremlin-Bicêtre, France
- ††Inserm U999, Laboratoire d'Excellence en Recherche sur le Médicament et l'Innovation Thérapeutique (LabEx LERMIT), Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France
- ‡‡Service d'Anatomie Pathologique, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France
- §§Service de Chirurgie Thoracique, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France
- ‖‖ICIN Netherlands Heart Institute, Utrecht, the Netherlands
- ↵∗Reprint requests and correspondence:
Dr. Frances S. de Man, VU University Medical Center, Department of Pulmonary Diseases, Boelelaan 1017, 1081 HV Amsterdam, the Netherlands.
- ↵∗∗Dr. Coen A. C. Ottenheijm, VU University Medical Center, Department of Physiology, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands.
Background After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function.
Objectives The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients.
Methods LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated.
Results Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (∼30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (∼25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ∼30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation.
Conclusions The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients.
- contractile protein phosphorylation
- left ventricular dysfunction
- myocyte physiology
- pulmonary arterial hypertension
Left ventricular (LV) failure often occurs as a post-operative complication after lung transplantation in patients with pulmonary arterial hypertension (PAH) (1–4). It is associated with increased duration of intensive care stay and post-operative mortality (5,6). Recent studies suggest that LV dysfunction already occurs in early stages of PAH (7–10). LV dysfunction in PAH patients is characterized by reduced ejection fraction (EF) and impaired diastolic function, and some studies reported a reduction in LV free wall mass in PAH patients (7,9,11,12). However, the pathophysiology of LV dysfunction in PAH is incompletely understood.
Important determinants of LV function are cardiomyocyte cross-sectional area (CSA) and contractility. Recent data from pulmonary hypertension rats show that LV cardiomyocyte CSA is reduced in end-stage disease (13–15). However, it is unknown whether atrophy of LV cardiomyocytes also occurs in humans with PAH. The contractility of cardiomyocytes largely depends on the functioning of the contractile apparatus: the sarcomeres. The force-generating capacity of sarcomeres is tightly regulated by post-translational modifications of sarcomeric proteins and by myosin-actin cross-bridge interactions. Whether sarcomere contractility is altered in LV cardiomyocytes of PAH patients is currently unknown.
The aim of the present study was to investigate the CSA and contractility of LV cardiomyocytes isolated from biopsies of PAH patients.
A more detailed description of the materials and methods is available in the Online Appendix.
Cardiac magnetic resonance imaging (MRI) was performed in 14 PAH patients and 15 control subjects as described previously (16). End-stage PAH patients were selected based on the availability of cardiac MRI within 3 to 17 months (median, 10 months) before heart/lung transplantation or death.
Left ventricular biopsies
LV free wall tissue of 9 end-stage PAH patients was collected during heart/lung transplantation. Nonfailing LV tissue was obtained from 10 donor hearts. Samples were obtained after informed consent and with the approval of the local ethical committee.
Cardiomyocyte CSA was determined in 5-μm cryosections stained with a laminin antibody (1:200, L9393, Sigma-Aldrich, Zwijndrecht, the Netherlands) (17,18). Sarcomere length was measured in perpendicular cryosections stained with anti–alpha-actinin (1:40, A7811, Sigma Aldrich), and CSA was normalized to a sarcomere length of 2.0 μm (see Online Fig. 1).
Contractile function of LV cardiomyocytes
To study sarcomere contractility, a single permeabilized LV cardiomyocyte of a PAH patient or donor was attached between a force transducer and a length motor, as described previously (19–21). To determine tension, force was normalized to cardiomyocyte CSA. In addition, we measured the rate of force redevelopment (κtr) and the Ca2+ sensitivity of force (22).
Active stiffness was determined in a subset of cardiomyocytes by placing the cardiomyocyte in activating solution, when steady isometric tension was reached. Small length perturbations (1.4%, 2.1%, and 2.8% of initial length) were imposed on the cardiomyocyte, resulting in a quick force response (Fig. 1). The tension change (ΔT) was plotted as a function of the length change (ΔL) (23).
Phosphorylation of sarcomeric proteins
To investigate post-translational modifications of sarcomeric proteins, ProQ Diamond stained 1-dimensional gels and Western blotting were used as described previously (20). In addition, myosin/actin ratio was calculated using SYPRO-stained gels.
Cardiac MRI measurements, CSA, phosphorylation, and protein content of the sarcomeric proteins were analyzed by independent Student t test or Mann-Whitney U test for non-normally distributed values. Two-way repeated measure analysis of variance was used to analyze the contractile parameters with Bonferroni post hoc tests. A p value of <0.05 was considered significant.
Patients' characteristics, hemodynamics, and MR results are shown in Table 1. The right ventricle (RV) of PAH patients was dilated and hypertrophied, which was accompanied by a decrease in RVEF. LVEF was significantly reduced in PAH patients, and a trend (p = 0.055) toward a lower LV end-diastolic volume was observed. Total LV mass index was increased in PAH patients; however, after excluding the interventricular septum, LV free wall mass index was not significantly different between the groups.
Atrophy of LV cardiomyocytes
An overview of the patients' demographics is shown in Table 2. No statistical differences were observed between the donors and PAH patients, with regard to age or sex.
Cardiomyocyte CSA was determined in laminin-stained LV cryosections. (For a typical example, see Fig. 2A.) LV cardiomyocyte CSA was significantly lower (∼30%) in PAH patients (Fig. 2B). To evaluate contractile protein content, the myosin/actin ratio was determined. A significant reduction (∼30%) in the myosin/actin ratio was found in PAH patients (Fig. 2C). Note that myosin/desmin ratio was reduced by 55% in PAH patients, whereas actin/desmin ratio was only reduced by 25%. This indicates that LV cardiomyocytes are not only smaller, but they also contain less myosin than donor cardiomyocytes do.
Depressed contractility of LV cardiomyocytes
The force-generating capacity—normalized to CSA (e.g., tension)—was determined in permeabilized single LV cardiomyocytes of PAH patients and donors. As in vivo cardiomyocytes operate at a range of sarcomere lengths, tension was determined at sarcomere lengths of 1.8, 2.0, and 2.2 μm (Fig. 3A). LV cardiomyocytes of both PAH patients and donors showed an increase in maximal tension with increasing sarcomere length, indicating preserved length dependence of maximal force in PAH. However, across the whole range of sarcomere lengths, maximal tension was significantly lower (25% to 27%) in LV cardiomyocytes of PAH patients (pinteraction <0.05). This indicates that contractile strength of LV cardiomyocytes is severely reduced in PAH patients.
Cross-bridge cycling kinetics
To evaluate the underlying cause of the reduced maximal tension, additional experiments were performed. The active force generated by sarcomeres in permeabilized, single cardiomyocytes is determined by: 1) the fraction of strongly bound cross bridges; 2) the number of available cross bridges; and 3) the force per cross bridge (24,25). A reduction in maximal tension should be accompanied by a change in 1 or more of these 3 determinants.
The κtr provides information on the attachment and detachment rate of the cross bridges during isometric contraction and reflects the fraction of strongly bound cross bridges. No change in κtr was observed between cardiomyocytes from PAH patients and donors (donor vs. PAH, 1.8 μm: 0.71 ± 0.03 [s-1] vs. 0.72 ± 0.05 [s-1]; 2.0 μm: 0.65 ± 0.03 [s-1] vs. 0.63 ± 0.03 [s-1]; and 2.2 μm: 0.66 ± 0.05 [s-1] vs. 0.58 ± 0.04 [s-1]), suggesting that changes in the fraction of strongly bound cross bridges do not contribute to the reduced maximal tension.
By measuring active stiffness, an estimate of the number of cross bridges during activation can be obtained. (For details, see Fig. 1.) Active stiffness was significantly reduced in PAH cardiomyocytes at a sarcomere length of 2.2 μm (Fig. 3B). This finding suggests that the number of available cross bridges is reduced in PAH cardiomyocytes, which is further supported by the observed reduction in myosin/actin ratio (Fig. 2C). The tension/stiffness ratio, which reflects the force generated per cross bridge, was not significantly different (Fig. 3C). Together, these results suggest that the reduced tension of LV cardiomyocytes in PAH patients is predominantly caused by a reduction in the number of available cross bridges.
Increased calcium sensitivity of force in LV cardiomyocytes
We also determined the Ca2+ sensitivity of force in LV cardiomyocytes by exposing them to solutions with incremental [Ca2+] and measuring the force response. A significant leftward shift in the normalized force [Ca2+] relation was observed at both 1.8 and 2.2 μm sarcomere lengths (Figs. 4A and 4B), indicating an increase in the Ca2+ sensitivity of force in LV cardiomyocytes of PAH patients. The [Ca2+] at which 50% of the force is reached (EC50) was determined in all cells. A significant decrease in EC50 was found at both 1.8 and 2.2 μm in PAH patients (EC50 donor vs. PAH, 1.8 μm: 6.12 ± 0.21 μmol/l vs. 5.07 ± 0.34 μmol/l; 2.2 μm: 5.49 ± 0.26 μmol/l vs. 4.22 ± 0.21 μmol/l), indicating that LV cardiomyocytes from PAH patients require less calcium for the same force response than donors do.
At submaximal Ca2+ concentrations, the increased Ca2+ sensitivity of force in LV cardiomyocytes of PAH patients partially compensates for the reduced force-generating capacity, which is visualized in Figures 4C and 4D by plotting the tension [Ca2+] relation. Active tension is significantly lower in cardiomyocytes from PAH patients than in donors at sarcomere lengths of 1.8 μm and 2.2 μm at [Ca2+] higher than 10 and 6.3μmol/l, respectively.
Unaltered diastolic stiffness of LV cardiomyocytes
Finally, we determined the passive tension of cardiomyocytes as a reflection of LV diastolic stiffness. No difference in passive tension was observed between groups at all sarcomere lengths (donor vs. PAH, 1.8 μm: 0.95 ± 0.12 kN/m2 vs. 1.30 ± 0.35 kN/m2; 2.0 μm: 1.57 ± 0.17 kN/m2 vs. 1.88 ± 0.41 kN/m2; and 2.2 μm: 2.42 ± 0.38 kN/m2 vs. 2.67 ± 0.68 kN/m2), suggesting no change in LV diastolic stiffness.
Hypophosphorylation of contractile proteins
To investigate the potential molecular mechanisms that underlie the reduced tension and increased Ca2+ sensitivity of LV cardiomyocytes in PAH patients, we assessed the phosphorylation status of sarcomeric proteins (20,26).
Compared with donor LV tissue, overall phosphorylation of cardiac myosin binding protein C (cMyBPC) and cardiac troponin I (cTnI) was significantly lower in the PAH patients (Fig. 5A). A reduction of 44 ± 6% (Fig. 5B) and 34 ± 6% (Fig. 5C), respectively, for cMyBPC and cTnI, was found in PAH patients. Phosphorylation of desmin and troponin T did not significantly differ between groups.
By Western blot analyses, we confirmed the increase of unphosphorylated cTnI in PAH patients compared with the donors. An antibody directed against unphosphorylated cTnI (22b11) showed a significant increase (219 ± 50%) in unphosphorylated cTnI in PAH patients (Fig. 5D).
By combining cardiomyocyte mechanics with analyses of cardiomyocyte structure and protein composition (Central Illustration), we demonstrate that LV cardiomyocytes from PAH patients have:
1. smaller CSA, indicating atrophy;
2. reduced force-generating capacity that persists after correction for atrophy, which is partly caused by loss of the major contractile protein myosin; and
3. reduced phosphorylation levels of key sarcomeric proteins, which may be responsible for the increased Ca2+ sensitivity of force.
We propose that these changes at the cardiomyocyte level contribute to the reduction of in vivo contractility of the LV in PAH patients, as indicated by our observations with MRI.
Cardiomyocyte atrophy and weakness contribute to LV dysfunction in PAH
Several studies have reported on impaired LV function in PAH patients (7,9–12). LV dysfunction in PAH is closely related to changes in RV function, either via direct ventricular interaction, which in PAH patients leads to limitations in LV diastolic filling by leftward septum bowing, or via hemodynamic effects as a result of a decrease in RV output that causes reduced filling of the LV (7,9,27).
Thus far, the pathophysiology of LV dysfunction has not been completely understood. Using MRI, a reduced LV pump function in PAH patients was observed, with no change in LV free wall mass. Some previous studies have demonstrated a higher total LV mass in PAH patients, but they did not report that the LV free wall mass separated from the interventricular septum (7), whereas others have reported a significant reduction of LV free wall mass in PAH patients (11). Importantly, using biopsy material, we observed a marked reduction in the CSA of individual LV cardiomyocytes in PAH patients, indicating that these cardiomyocytes are atrophied. The current study is the first to investigate the CSA of LV cardiomyocytes in PAH patients. However, our clinical findings are supported by data from several studies in PAH rats, which also reported a reduction of LV cardiomyocyte CSA (13–15). The combination of reduced LV cardiomyocyte CSA with unaltered LV free wall mass suggests that extracellular changes (i.e., increased fibrosis or edema) may occur in the LV of PAH patients. Indeed, previous work indicated increased collagen content and fibrosis in the LV of PAH rats (14,15,28).
The combined reduction in CSA and contractile function of cardiomyocytes leads to a substantial decrease in the pressure-generating capacity of the LV. Together, these findings indicate that intrinsic alterations in cardiomyocytes impact LV systolic function in PAH patients and contribute to the reduction in LVEF (7,9,11) and LV strain in these patients (10).
In addition to systolic dysfunction, LV diastolic function and LV relaxation are impaired in PAH patients (12,29). Interestingly, we did not observe a change in cardiomyocyte stiffness in PAH patients. Therefore, we speculate that it is the increase in myofilament Ca2+ sensitivity in PAH cardiomyocytes that contributes to LV diastolic dysfunction in PAH, not changes in passive stiffness of LV cardiomyocytes. It should be noted that extrasarcomeric changes, such as calcium handling, by the sarcoplasmic reticulum (e.g., changes in sarcoplasmic/endoplasmic reticulum calcium ATPase expression, phospholamban phosphorylation) also contribute to LV diastolic dysfunction in PAH patients (30). Such changes could potentially affect both the contractility and relaxation kinetics of the LV.
LV adaptations in PAH: caused by reduced filling?
In PAH patients, LV filling is reduced due to impaired RV output and to septum bulging. We propose that reduced LV filling, and the concomitant reduced volume load of the LV, underlie the changes in LV cardiomyocytes in PAH patients. Previous work showed that sustained reductions in cardiac load, induced by spaceflight or bed rest, lead to reductions in LV mass (31–33) and cardiomyocyte CSA (30). These unloading-induced changes are similar to the changes observed in the LV cardiomyocytes of PAH patients. Furthermore, in unloaded rat hearts, induced by heterotopic heart transplantation to the abdominal aorta, the contractile reserve, and Ca2+ regulation of intact rat cardiomyocytes of unloaded hearts were depressed, suggesting cardiomyocyte contractile dysfunction (30). Finally, unloading of the heart in rats has been shown to reduce phosphorylation levels of cTnI (26), a finding that mimics the reduction in cTnI phosphorylation observed in LV cardiomyocytes of PAH patients (Fig. 5).
Additional support for the notion that atrophy and weakness of LV cardiomyocytes are a consequence of LV unloading is provided by the comparison of LV cardiomyocytes with those of RV cardiomyocytes of the same PAH patients. In contrast to LV cardiomyocytes, RV cardiomyocytes are overloaded as a result of the increased pulmonary vascular resistance. Hence, opposite findings in RV cardiomyocytes are expected if changes in loading explain the reduction in LV cardiomyocyte size and function. Recently, our group published findings on RV cardiomyocyte function in PAH patients (18). Importantly, the LV cardiomyocytes studied here are derived from mostly the same individuals from whom RV cardiomyocytes were studied. Indeed, opposite to the LV cardiomyocytes, we observed an increase in the force-generating capacity and cardiomyocyte size in the RV of PAH patients compared with donors. Furthermore, a 3-fold increase in RV cardiomyocyte stiffness was found in PAH patients, whereas stiffness was not changed in LV cardiomyocytes from PAH patients (18). The opposing results from LV and RV cardiomyocytes from the same patients strongly support our proposition that unloading of the LV—rather than systemic effects associated with PAH—underlie the changes in contractile function of LV cardiomyocytes.
The group of PAH patients consisted of patients with idiopathic PAH (n = 3) and congenital heart disease-associated PAH (n = 6). It could be speculated that these different causes of PAH differentially affect LV function. Therefore, we also compared analyses of histology, contractility, and phosphorylation between idiopathic PAH and congenital heart disease patients. Comparable results for CSA, maximal tension, EC50, myosin/actin ratio, cTnI phosphorylation, and cMyBPC phosphorylation of LV cardiomyocytes were found in idiopathic PAH and congenital heart disease patients (Online Figs. 2 and 3), suggesting that the origin of PAH does not affect LV function in a different manner.
We aimed to increase our clinical understanding of the changes in the LV in PAH by studying in vivo LV function using MRI. As MRI data were not available from the PAH patients from whom biopsy specimens were obtained, measurements were performed in a separate cohort of PAH patients. Both PAH groups consisted of patients with end-stage disease, undergoing a heart/lung transplant or awaiting heart/lung transplantation. Therefore, we argue that both groups are comparable and that the data can be translated.
Control LV tissue was obtained from organ donors and, theoretically, high-circulating catecholamine levels before organ harvest have affected phosphorylation levels in LV tissue from the donors. However, phosphorylation of contractile proteins affects mostly the Ca2+ sensitivity of force in cardiomyocytes, with only minor or no effects on our key parameters: maximal active tension, passive tension, and cardiomyocyte CSA. It is, therefore, unlikely that differences between groups in these key parameters were caused by an altered phosphorylation status in donors. This notion is further supported when comparing these results with previous findings on the RV of the same subjects (18). Importantly, whereas both the LV and RV have been exposed to similar levels of systemic catecholamines, the changes in maximal and passive tension, and CSA in LV versus RV cardiomyocytes are in the opposite direction. This suggests that these changes are not caused by the phosphorylation status but are driven by other factors.
Finally, it has been shown that cardiomyocyte contractility differs between various locations in the LV free wall (34). The precise locations of the LV biopsies are unknown, but LV biopsies are routinely taken from random locations in the free wall of the LV. Furthermore, measurements were performed on random pieces of tissue isolated from the biopsies. Therefore, all biopsies and biopsy pieces together are likely to be representative for the LV as a whole.
The present findings provide new insights into the mechanisms underlying LV dysfunction in PAH. We demonstrate that LV cardiomyocytes of PAH patients are not only atrophied but also have a severely impaired contractility. We propose that contractile dysfunction of LV cardiomyocytes contributes to the post-operative complications observed in PAH patients after a lung transplant, when the LV is suddenly re-exposed to higher filling pressures. Future therapeutic strategies aimed at targeting the contractile impairment and atrophy will reveal the clinical implications of LV dysfunction in PAH patients.
COMPETENCY IN MEDICAL KNOWLEDGE: In patients with PAH, LV cardiomyocytes are atrophic, and cardiomyocyte contractile function is impaired. This can explain the reduction of in vivo contractility observed in the LV of PAH patients.
TRANSLATIONAL OUTLOOK 1: Measuring LV function in PAH patients prior to lung transplant might predict post-operative cardiac complications but requires further investigation.
TRANSLATIONAL OUTLOOK 2: Future therapeutic strategies aimed at targeting the contractile impairment and atrophy will reveal the clinical implications of LV dysfunction in patients with PAH.
The authors thank Max Goebel, Pim Stam, and Silvia Rain for their technical assistance.
For supplemental methods as well as figures, please see the online version of this article.
Supported by VENI (#916.14.099) and VIDI (#917.12.319; #917.11.344; #917.96.306) grants from the Dutch Foundation for Scientific Research awarded to Drs. van der Velden, Vonk-Noordegraaf, de Man, and Ottenheijm. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. de Man and Ottenheijm contributed equally to this work.
- Abbreviations and Acronyms
- cross-sectional area
- cardiac troponin I
- cardiac myosin binding protein C
- ejection fraction
- left ventricle/ventricular
- magnetic resonance imaging
- pulmonary arterial hypertension
- right ventricle
- Received January 27, 2014.
- Accepted April 16, 2014.
- American College of Cardiology Foundation
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