Multimodality assessment of heart failure with preserved ejection fraction skeletal muscle reveals differences in the machinery of energy fuel metabolism

Payman Zamani, Elizabeth A Proto, Neil Wilson, Hossein Fazelinia, Hua Ding, Lynn A Spruce, Antonio Davila Jr, Thomas C Hanff, Jeremy A Mazurek, Stuart B Prenner, Benoit Desjardins, Kenneth B Margulies, Daniel P Kelly, Zoltan Arany, Paschalis-Thomas Doulias, John W Elrod, Mitchell E Allen, Shana E McCormack, Gayatri Maria Schur, Kevin D'Aquilla, Dushyant Kumar, Deepa Thakuri, Karthik Prabhakaran, Michael C Langham, David C Poole, Steven H Seeholzer, Ravinder Reddy, Harry Ischiropoulos, Julio A Chirinos, Payman Zamani, Elizabeth A Proto, Neil Wilson, Hossein Fazelinia, Hua Ding, Lynn A Spruce, Antonio Davila Jr, Thomas C Hanff, Jeremy A Mazurek, Stuart B Prenner, Benoit Desjardins, Kenneth B Margulies, Daniel P Kelly, Zoltan Arany, Paschalis-Thomas Doulias, John W Elrod, Mitchell E Allen, Shana E McCormack, Gayatri Maria Schur, Kevin D'Aquilla, Dushyant Kumar, Deepa Thakuri, Karthik Prabhakaran, Michael C Langham, David C Poole, Steven H Seeholzer, Ravinder Reddy, Harry Ischiropoulos, Julio A Chirinos

Abstract

Aims: Skeletal muscle (SkM) abnormalities may impact exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF). We sought to quantify differences in SkM oxidative phosphorylation capacity (OxPhos), fibre composition, and the SkM proteome between HFpEF, hypertensive (HTN), and healthy participants.

Methods and results: Fifty-nine subjects (20 healthy, 19 HTN, and 20 HFpEF) performed a maximal-effort cardiopulmonary exercise test to define peak oxygen consumption (VO2, peak ), ventilatory threshold (VT), and VO2 efficiency (ratio of total work performed to O2 consumed). SkM OxPhos was assessed using Creatine Chemical-Exchange Saturation Transfer (CrCEST, n = 51), which quantifies unphosphorylated Cr, before and after plantar flexion exercise. The half-time of Cr recovery (t1/2, Cr ) was taken as a metric of in vivo SkM OxPhos. In a subset of subjects (healthy = 13, HTN = 9, and HFpEF = 12), percutaneous biopsy of the vastus lateralis was performed for myofibre typing, mitochondrial morphology, and proteomic and phosphoproteomic analysis. HFpEF subjects demonstrated lower VO2,peak , VT, and VO2 efficiency than either control group (all P < 0.05). The t1/2, Cr was significantly longer in HFpEF (P = 0.005), indicative of impaired SkM OxPhos, and correlated with cycle ergometry exercise parameters. HFpEF SkM contained fewer Type I myofibres (P = 0.003). Proteomic analyses demonstrated (a) reduced levels of proteins related to OxPhos that correlated with exercise capacity and (b) reduced ERK signalling in HFpEF.

Conclusions: Heart failure with preserved ejection fraction patients demonstrate impaired functional capacity and SkM OxPhos. Reductions in the proportions of Type I myofibres, proteins required for OxPhos, and altered phosphorylation signalling in the SkM may contribute to exercise intolerance in HFpEF.

Keywords: Exercise; HFpEF; Skeletal muscle.

Conflict of interest statement

Dr. Zamani has consulted for Vyaire (modest). Dr. Mazurek has received advisory board honoraria from Actelion Pharmaceuticals (modest) and United Therapeutics (modest). Dr. Margulies receives research funding from Sanofi‐Aventis (significant), Merck (significant), and GlaxoSmithKline (significant). Dr. Kelly received advisory board honoraria from Pfizer (significant) and Amgen (modest). Dr. Ischiropoulos is the Gisela and Dennis Alter endowed chair. Dr. Chirinos has received consulting honoraria from Sanifit (significant), Microsoft (modest), Fukuda‐Denshi (modest), Bristol‐Myers Squibb (modest), OPKO Healthcare (modest), Ironwood Pharmaceuticals (modest), Pfizer (modest), Akros Pharma (modest), Merck (modest), Edwards Lifesciences (modest), and Bayer (significant). Dr. Chirinos has received research grants from Microsoft, Fukuda‐Denshi and Bristol‐Myers Squibb (all significant). He is named as inventor in an UPenn patent for the use of inorganic nitrates/nitrites for the treatment of HFpEF and a patent application for the use of novel neoepitope biomarkers of tissue fibrosis in heart failure.

© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.

Figures

Figure 1
Figure 1
Creatine chemical exchange saturation transfer (CrCEST) maps of exercise‐induced changes in skeletal muscle creatine (Cr) concentration. Data displayed are from one healthy 36‐year‐old male following 2 min of plantar flexion exercise against 7 PSI pressure at 0.75 Hz. Top panel: CrCEST maps before (enclosed by white box) and after exercise show an increase in CrCEST asymmetry (increase in red shading), indicative of increased free Cr, liberated as part of the phosphocreatine shuttle to generate ATP (PCr+ADP ↔ Cr+ATP). During recovery, free Cr decreases as the ATP produced by oxidative phosphorylation shifts the reaction towards PCr regeneration. Bottom panel: Changes in CrCEST asymmetry before and after exercise are highlighted in prescribed regions of interest: lateral gastrocnemius (green), medial gastrocnemius (red), and the soleus (blue) muscles. Data are displayed as mean ± SD at each time point. The mean at each time point represents the average value from all activated voxels, and its standard deviation, from within each muscle.
Figure 2
Figure 2
Group data for key endpoints. Data are presented as mean with 95% confidence intervals. When the data were not normally distributed, log‐transformation was performed for statistical analyses, and geometric means with their 95% confidence intervals are plotted to retain native units. ANOVA was used for the overall comparison, with post hoc intergroup comparisons performed with Bonferroni correction. *Adjusted P < 0.05, **adjusted P ≤ 0.01, ***adjusted P ≤ 0.001.
Figure 3
Figure 3
Volcano plot, network map, and biologic enrichment of proteins significantly different between HFpEF and HTN participants. (A) Volcano plots were constructed with blue dots representing proteins with significantly different relative levels between HFpEF and HTN participants (P < 0.05), and red dots and the gene names listed for proteins that had significantly different levels with an absolute log2 fold‐change (FC) > 1. (B) All proteins with significantly different relative levels between HFpEF and HTN participants (P < 0.05), and their log2 fold‐change (FC), were entered into the String Database (string-db.org). Interrelated proteins are displayed along with the connection between proteins and groups. Related proteins are shaded a similar colour. The halo around each protein represents the log2 FC, with blue indicating a relative decrease in protein level in HFpEF participants as compared with HTN participants, and red representing an increase. Although not inclusive, specific clusters of proteins related to energy fuel metabolism are enumerated. (C) Enrichment of the top 5 biologic (GO) processes, along with the false‐discovery rate are listed. AKGDH, alpha‐ketoglutarate dehydrogenase complex; BCKDH, branched‐chain alpha‐keto dehydrogenase complex; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid cycle.
Figure 4
Figure 4
Volcano plot and network map of proteins significantly different between HFpEF and healthy participants. (A) Volcano plots were constructed with blue dots representing proteins with significantly different relative levels between HFpEF and healthy participants (P < 0.05), and red dots and the gene names of proteins that had significantly different levels with an absolute log2 fold‐change (FC) > 1. (B) All proteins with significantly different relative levels between HFpEF and healthy participants (P < 0.05), and their log2 fold‐change (FC), were entered into the String Database (string-db.org). Interrelated proteins are displayed, along with the connection between proteins and groups. Related proteins are shaded a similar colour. The halo around each protein represents the log2 FC, with blue indicating a relative decrease in protein level in HFpEF participants as compared with healthy participants, and red representing an increase. Although not inclusive, specific clusters of proteins have been enumerated. (C) Enrichment of the top biologic (GO) processes and the false‐discovery rate are listed. AKGDH, alpha‐ketoglutarate dehydrogenase complex; BCAA, branched chain amino acids; BCKDH, branched‐chain alpha‐keto dehydrogenase complex; PDH, pyruvate dehydrogenase, TCA, tricarboxylic acid cycle.
Figure 5
Figure 5
HFpEF vs. HTN muscle biopsy samples demonstrate relative differences in the phosphoproteome. The volcano plot (A) compares the differences in the relative levels of specific phosphopeptides, identifying differences in 522 phosphopeptides (P < 0.05 for each). Blue dots represent peptides with significantly different relative levels (P < 0.05); whereas, red dots indicate peptides with significantly different relative levels and an absolute log2 fold‐change >1. Peptides and the site of phosphorylation are listed. In (B), kinase motif analysis using the human phosphopeptide proteome as a reference identified increased enrichment for a P‐X‐(S/T)‐P motif in HTN as compared with HFpEF SkM. This motif is recognized by ERK1/ERK2, among other kinases.

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