Right Ventricular Myofilament Functional Differences in Humans With Systemic Sclerosis-Associated Versus Idiopathic Pulmonary Arterial Hypertension

Abstract

Background: Patients with systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) have a far worse prognosis than those with idiopathic PAH (IPAH). In the intact heart, SSc-PAH exhibits depressed rest and reserve right ventricular (RV) contractility compared with IPAH. We tested whether this disparity involves underlying differences in myofilament function.

Methods: Cardiac myocytes were isolated from RV septal endomyocardial biopsies from patients with SSc-PAH, IPAH, or SSc with exertional dyspnea but no resting PAH (SSc-d); control RV septal tissue was obtained from nondiseased donor hearts (6-7 per group). Isolated myocyte passive length-tension and developed tension-calcium relationships were determined and correlated with in vivo RV function and reserve. RV septal fibrosis was also examined.

Results: Myocyte passive stiffness from length-tension relations was similarly increased in IPAH and SSc-PAH compared with control, although SSc-PAH biopsies had more interstitial fibrosis. More striking disparities were found between active force-calcium relations. Compared with controls, maximal calcium-activated force (F_max) was 28% higher in IPAH but 37% lower in SSc-PAH. F_max in SSc-d was intermediate between control and SSc-PAH. The calcium concentration required for half-maximal force (EC₅₀) was similar between control, IPAH, and SSc-d but lower in SSc-PAH. This disparity disappeared in myocytes incubated with the active catalytic subunit of protein kinase A. Myocyte F_max directly correlated with in vivo RV contractility assessed by end-systolic elastance (R² =0.46, P=0.002) and change in end-systolic elastance with exercise (R² =0.49, P=0.008) and was inversely related with exercise-induced chamber dilation (R² =0.63, P<0.002), which also was a marker of depressed contractile reserve.

Conclusions: A primary defect in human SSc-PAH resides in depressed sarcomere function, whereas this is enhanced in IPAH. These disparities correlate with in vivo RV contractility and contractile reserve and are consistent with worse clinical outcomes in SSc-PAH. The existence of sarcomere disease before the development of resting PAH in patients with SSc-d suggests that earlier identification and intervention may prove useful.

Keywords: heart ventricles; hypertension, pulmonary; myofibrils; protein kinases; scleroderma, systemic.

Figure 1

Effects of SSc-PAH versus IPAH on passive myocyte tension and fibrosis (A) Representative Masson Trichrome histologic images from IPAH, SSc-PAH, and SSc. (B) Histologic fibrosis measured by Masson Trichrome as a percentage of total biopsy area was increased in SSc-PAH versus IPAH or SSc-d, based on a threshold of 2.5% fibrosis. (C) Passive tension as a function of escalating sarcomere length was measured in IPAH, SSc-PAH, and non-failing control myocytes (n=5 subjects/group). Passive tension between all three groups were compared using one-way analysis of variance (ANOVA) at each SL. * post-hoc P<0.05 between Control and IPAH; ^† post-hoc P<0.05 between Control and SSc-PAH.

Figure 2

Force-Calcium curves in Control, IPAH, and SSc myofilaments (A) Force-calcium data were obtained in non-failing control, IPAH, and SSc-PAH myocytes to obtain maximal calcium-activated tension (F_max) and calcium sensitivity (EC₅₀) (n=7 subjects/group). (B) Data were normalized to maximal tension to illustrate shifts in calcium sensitivity (measured as EC₅₀, or the calcium concentration necessary for 50% maximal activation). (C) Myofilament force-calcium data of control, SSc-PAH, and SSc subjects free of PAH (SSc-d). (D) F_max, EC₅₀, Hill coefficient (h), and myocyte cross-sectional area (CSA) data; data presented as mean ± SD. F_max was increased in IPAH versus controls, whereas it was decreased in SSc-PAH versus both control and IPAH; in SSc-d, F_max was intermediate between control and SSc-PAH. EC₅₀ was decreased in SSc-PAH versus control, IPAH, and SSc-d. There was no significant difference between groups in Hill coefficient or myocyte cross-sectional area (CSA). * significant difference in post-hoc comparison.

Figure 3

Effect of PKA treatment on myofilament function and passive myocyte stiffness (A) Myofilaments from a subset of control, IPAH, and SSc-PAH subjects (3 subjects/group) were directly incubated with protein kinase A (PKA). (B) Baseline calcium sensitivity (EC₅₀) in all three groups mirrored trends of the larger cohort. With PKA, there was a slight increase in EC₅₀ in control and IPAH. On the other hand, PKA completely markedly increased EC₅₀ in the SSc-PAH group to control levels. Change in EC₅₀ was significantly greater in SSc-PAH than in both control and IPAH (Disease × group interaction P=0.002). (C) PKA treatment led to a decrease in passive tension, at a sarcomere length (SL) of 2.1 μm, in both IPAH and SSc-PAH, but no change in controls. There was no significant difference in PKA-mediated passive tension change between IPAH and SSc-PAH. * P<0.05; post-hoc P-value applied where applicable.

Figure 4

Myofilament Fmax correlates with in vivo RV function and functional reserve during exercise (A) Myofilament F_max correlated strongly with resting RV chamber-level contractility (Ees) (R²=0.46, P=0.002). (B) F_max also correlated strongly with contractile reserve, or change in Ees with exercise (R2=0.49, P=0.008), as well as exercise RV chamber dilation, as measured by percent change in RV end-diastolic volume (RV EDV) after reaching a 25-Watt workload (R²=0.63, P<0.002).