Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy

Abstract

Background

Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM.

Methods

We performed a comprehensive multi-omics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts).

Results

Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites [ATP, ADP, and phosphocreatine (PCr)] and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase (CS) activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species (ROS) and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance.

Conclusions

Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

INTRODUCTION

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease, affecting 1 in 500 individuals, and a leading cause of heart failure and sudden death^1–4. HCM is characterized by often asymmetric thickening of the left ventricle (LV), fibrosis and reduced diastolic function. Systolic function is usually hyperdynamic initially, but can progress to systolic dysfunction and clinical heart failure⁵. Additional clinical phenotypes include LV outflow tract (LVOT) obstruction and arrhythmia^{6, 7.} To date, no treatment has been shown to slow disease progression, although mavacamten has shown promise in reducing LV hypertrophy⁸. Advances in genetics over the past two decades have uncovered over 1,000 mutations associated with HCM most often in sarcomeric genes such as cardiac myosin-binding protein-C (MYBPC3) and β-cardiac myosin (MYH7)^{3, 9}. Biochemical and biophysical studies of isolated human cardiac myosin suggest that different HCM mutations can have heterogeneous effects on actin gliding velocity, intrinsic force, and ATPase activity^{3, 10, 11}. At the cellular level, most HCM mutations increase total force production and ATP utilization, resulting in higher myocardial energy demand¹². Inefficient energy utilization for force generation has been suggested as a primary biophysical consequence of HCM sarcomeric mutations, leading to energetic stress and adverse remodeling^12–15,16. The normal heart has an extreme degree of metabolic flexibility, utilizing various substrates to meet changes in energy demand⁴. Alterations in cardiac substrate utilization have been associated with compromised energy supply in HCM^17–21 including lipid metabolism and mitochondrial oxidative phosphorylation²¹. Of note, patients with mutations in mitochondrial DNA often develop an HCM phenotype, indicating that primary energetic alterations can also lead to HCM pathology^{22, 23}. Early electron micrographic studies demonstrated mitochondrial fission and disruption of cristae before their role in cardiac remodeling was fully understood^24–26. Impairment of mitochondrial function and morphology have more recently been described in murine models^{20, 27} and in HCM patients¹⁹. Despite evidence linking energy metabolism and HCM, the molecular mechanisms involved in the initiation and progression of HCM disease are still largely unknown as are the role of alterations in mitochondrial structure and function.

We performed multi-omics profiling of human myocardial tissue including metabolome, lipidome and transcriptome; ultrastructural tissue analysis using transmission electron microscopy (TEM); and mitochondrial functional studies. We uncovered a distinct metabolic profile in HCM, accompanied by markedly abnormal mitochondrial structure, respiratory dysfunction, and failure to upregulate mitophagic clearance, a normal mechanism for maintenance of mitochondrial integrity. These diffuse metabolic alterations could help identify new treatments targeted on rescuing progressive metabolic dysfunction and mitochondrial damage at an earlier stage of disease.

METHODS

The data that support the findings of the present study are available from the corresponding author upon reasonable request.

Study cohort and myocardial sample collection

HCM patients.

27 patients who underwent septal myectomy for clinical indications Inclusion criteria: normal or hyperdynamic LV function (ejection fraction≥55%) with a gradient across the LVOT (See Table 1 and Supplemental Materials online for details).

Table 1.

Characteristics of 42 subjects included in the cohort.

	Control		Disease
Group	Donor	Mitral Stenosis	HCM
NO. of patient samples	13	2	27
Female, n (%)	9 (69)	2 (100)	6 (22)
Age, year	50 ± 10	49 ± 6	54 ± 14
Positive Family Hx, n (%)			8 (30)
Mutations *
MYBPC3, n (%)			7 (28)
MYH7, n (%)			3 (12)
Others**,***, n (%)			2 (8)
VUS or no mutation, n (%)			13 (52)
Medication
β-blocker, n (%)			11 (41)
Calcium blocker, n (%)			19 (71)
Statin, n (%)			14 (52)
Echocardiogram
LVEF	58 ± 6	62 ± 6	67 ± 7
IVSd, cm			1.8 ± 0.5
LVPWd, cm			1.4 ± 0.4
IVSd/LVPWd ratio			1.4 ± 0.6
LVOT peak grad 40-60 mmHg, n (%)			2 (8)#
LVOT peak grad 60-90 mmHg, n (%)			4 (15)
LVOT peak grad > 90 mmHg, n (%)			20 (75)
LV Mass (ASE Method), g/m2; (LV mass index)			264 ± 100; (134 ± 45)##
Magnetic resonance imaging (MRI)
Delayed Enhancement (range)			1.5 ± 1.7 (0 - 4)
LV Mass, g/m2; (LV mass index)			187 ± 43; (94 ± 18)$

Continuous variables are expressed as mean ± SD and categorical variables as number (percentage); normal LV mass index²⁸ in men, 70.0 ± 8.9 g/m² and in women, 60.7 ± 8.1 g/m².

^*Genetic testing was performed on 25 patients.

^**one patient had two mutations.

^***mutations in TNNT, CSRP, and SCN.

^#in one patient with a resting gradient of 33 mmHg, exercise gradient was not available.

^##left ventricular mass on 24 patients calculated from echo data.

^$left ventricular mass on 9 patients calculated from MRI data.

HCM, hypertrophic cardiomyopathy; MYBPC, myosin binding protein C; MYH, myosin heavy chain; VUS, variants of unknown significance; LVEF, left ventricular ejection fraction; IVSd, interventricular septum diameter in diastole; LVPWd, left ventricular posterior wall thickness in diastole; LVOT peak grad, left ventricular outflow tract peak gradient; ASE, American Society of Echocardiography.

Controls.

Myocardial tissue was obtained from 13 donor hearts with no cardiac history, and 2 patients with primary mitral stenosis without significant insufficiency. The Stanford Institutional Review Board approved the study. All subjects provided written informed consent.

An overview of all techniques and data analysis is summarized in Figure 1A. A detailed method section including a list of software and reagents (Table I and Table II in the Supplement) is available in the Supplemental Materials online.

Figure 1. Study design and molecular profiles in HCM.

(A) Summary of the experimental design workflow. (B) Metabolomic, lipidomic, and transcriptomic profiling was performed on left ventricle tissue from HCM patients and donor controls. Principal component analysis (PCA) plots of all 6,189 metabolite features(left), 728 lipids (middle), and 48,167 transcripts (right) clearly separate the profiles of HCM from controls. (C) Top metabolic pathways enriched in HCM identified by IMPaLA, integrating metabolites, lipids, and transcripts (FDR<0.05). Metabolic pathways were categorized as follows: proteins, blue; lipids, orange; carbohydrates, black; nucleotides, gray and energy metabolism (purple). (D) Integrated network analysis combining all omic datasets using Shiny Genes and Metabolites (GAM) platform. Resulting networks were annotated and plotted in Cytoscape. Each node (circle) represents a metabolite or lipid and each edge connecting nodes represents an enzyme-encoding transcript based on biochemical relationships. The arrowhead determines the principal direction of the biochemical reaction. The size of each node and of connecting line reflects their p value; the color represents the relative change (blue, decrease; red, increase; black, not significant; and gray, not detected). n=6 control and n=13 HCM samples for metabolites/lipids; n=7 control and n=13 HCM for transcripts. FFA, free fatty acids; M/S-chain, medium/short-chain; L-chain, long chain; AC, acylcarnitine; G3P, glyceraldehyde 3-phosphate; PPP, pentose phosphate pathway; Ru5P, ribulose-5-phosphate; R5P, ribose 5-phosphate; CER, ceramide; CE, cholesterol ester; PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; DAG, diacylglycerol; and TAG, triacylglycerol.

Statistical Analysis

Data are shown as mean ± standard deviation (SD) or standard error (SE). Student’s t-test and Wilcoxon-Mann-Whitney U test were used for two-group comparisons of all non-omics data. P<0.05 was considered significant. For omics data, our statistical methods are described in the respective methods sections. Sample sizes are indicated in Figure 1A and figure legends. Pearson’s correlation coefficient and linear regression analyses were performed using pandas and scipy.

RESULTS

Cohort characteristics

LV septal myectomy samples were obtained from 27 patients with HCM: 22% women; age 54±14 years (mean±SD); and ejection fraction 67±7% (Table 1). Genetic testing was performed in 25 subjects. Twelve (48%) had a known or likely pathogenic HCM variants. Seven were identified in MYBPC3; three in MYH7; and one each in CSRP3, TNNT2, and SCN5A. Thirteen patients had one or more variants of unknown significance or no mutation detected. All patients had increased LV mass by both echocardiography and magnetic resonance imaging (MRI). Thirteen donor hearts were used as controls: 69% women; age, 50±10 years; and ejection fraction, 58±6%. Two additional patients, with mitral stenosis undergoing valve repair, were used as controls for mitochondrial respiration assays only: all women; age, 49±6 years; and ejection fraction, 62±6%. Clinical features of the study cohort are summarized in Table 1. Detailed clinical characteristics on HCM patients and controls are described in Table III and Table IV in the Supplement. Sample workflow and assay details are described in Table V in the Supplement.

Level of fibrosis was measured using gadolinium-enhanced cardiac MRI in 9 patients and was rated as mild (<4%) (Figure IA and Figure IB in the Supplement). However, given the limited resolution of MRI for detection of focal and interstitial fibrosis²⁹, we also performed histological analysis using hematoxylin/eosin and Masson Trichrome. HCM samples showed marked myocyte hypertrophy and increased interstitial collagen (%fibrosis HCM=11 vs. control=3.5) (Figure IC and Figure ID in the Supplement). We then analyzed the correlation between the degree of tissue fibrosis with the expression of fibrotic genes (ECM organization gene-set ; GO-0030198) and found only few genes were positively correlated (Excel I in the Supplement). Altogether, these data suggest that there is a mild/moderate level of fibrosis in septal myectomy samples.

Distinct molecular profiles discriminate HCM from controls

A multi-omics approach was used to characterize molecular composition of HCM (n=13) and control (n=6-7) myocardium as well as gaining insights into regulatory mechanisms. Metabolite and lipid profiling was performed using untargeted and targeted mass spectrometry and gene expression by RNA sequencing. A large proportion of metabolites and lipids were significantly altered (FDR<0.05; 40% (2,456/6,189) and 73% (530/728), respectively) (Figure II, Excel II, and Excel III in the Supplement), while 10% (5,033) of a total of 48,167 genes were differentially expressed in HCM (FDR<0.05) (Figure III in the Supplement). Principal component analysis (PCA) illustrates a clear separation of HCM from controls across omic datasets (Figure 1B).

Integrative omics reveals impaired energy metabolism in HCM

Integrated Molecular Pathway Level Analysis (IMPaLA)³⁰ was used to identify dysregulated pathways. Pathway enrichment revealed marked alterations in all metabolic pathways including lipids, amino acids, carbohydrates, and nucleotides suggesting a global dysregulation in energy metabolism (Figure 1C). A summary of the major metabolic changes was plotted on a metabolic network map (Figure 1D), demonstrating concordant alterations in the transcription of key metabolic enzymes and their associated metabolites or lipids.

In lipid metabolism, we observed a significant increase in free fatty acid (FFA) concentration (Figure 1D, Figure 2A, Figure IVA in the Supplement), despite reduced expression of the genes involved in their transport across the plasma membrane (SLC27A4, FABP4, and CD36) (Figure 2B, Figure IVB in the Supplement). After entering the cells, FFA are converted to acyl-CoAs and then acylcarnitine (AC) to enable entry into the mitochondria and ß-oxidization⁴. HCM myocardium showed a decrease in the abundance of all ACs, with long-chain ACs (>C14) the most impacted (Figure 1D, Figure 2C). This suggests a defect in conversion of FFA to AC, consistent with the marked reduction in free carnitine along with decreased trimethyllysine, the precursor of free carnitine (Figure 1D, Figure 2D). Consistent with this, mitochondrial carnitine acetyltransferase (CRAT), which catalyzes the conversion of acyl-CoA to Acetyl-carnitine using free carnitine, was reduced (Figure 1D). Additionally, genes associated with transporting ACs across the mitochondrial membrane (SLC25A, and SLC22A5) and carnitine palmitoyltransferase I (CPT1) which facilitate their transport were reduced in HCM (Figure 1D, Figure 2B, Figure IVB and Figure IVC in the Supplement). Importantly, the expression of genes encoding key enzymes involved in fatty acid β-oxidation were all reduced including: ACADVL, responsible for the first step of long-chain fatty acid oxidation; ACSL1, converting long-chain fatty acids to their active acyl-CoAs; HADHA and HADHB, catalyzing the last three steps of long-chain fatty acid oxidation; and ECH1, the final step of fatty acid oxidation to produce acetyl CoA (Figure 1D, Figure 2B). Besides entering mitochondria for oxidation, acyl-CoAs can also enter the de novo sphingolipid synthesis pathway to form ceramide (CER) and sphingomyelin (SM) which were both elevated in HCM (Figure 1D, Figure 2E). Similarly, there were significant increases in several phospholipids and lysophospholipids including cholesterol esters (CE), phosphatidylcholines (PC), phosphatidylethanolamines (PE), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE) (Figure 1D, Figure IVD–F in the Supplement). Despite the normal heart’s preference for lipids as an energy substrate, several studies have shown pathological effects of lipid overload in the heart, especially CER³¹. Triacylglycerol (TAG), the main source of lipid storage, however, was lower in HCM, with no changes in diacylglycerol (DAG), suggesting that overall energy storage (likely as lipid droplets) was lower (Figure 1D, Figure IVG in the Supplement). Consistent with reduced TAG levels, lipoprotein lipase (LPL), which hydrolyzes TAG for fatty acid generation, was significantly increased, along with downregulation of DGAT1, which forms TAG from DAG for energy storage (Figure 1D, Figure IVH in the Supplement).

Figure 2. Dysregulated metabolism in HCM.

(A) Total concentration (log₂ scale) of free fatty acids (FFA) in HCM as measured by the targeted lipidomics platform. (B) Heatmap shows overall changes in genes regulating myocardial energetics. Shown are expression of genes encoding major fatty acid transporters, β-oxidation enzymes, a glucose transporter, and enzymes involved in regulation of glycolysis, TCA cycle, and mitochondrial energy metabolism. (C-F) Levels of acylcarnitines (total concentration, log₂ scale), carnitine, sphingolipids (total concentration), and TCA cycle metabolites measured by untargeted metabolomics. (G) Citrate synthase (CS) activity in HCM vs control (spectrophotometry analysis). (H) High energy phosphate metabolites measured by untargeted metabolomics. Error bars represent mean ± SEM. n=6 control and n=13 HCM in A, C-F, and H; n=7 control and n=13 HCM in B; n=5 control and n=5 HCM in G. Between-group comparisons performed using Whitney U test in A, C, E, and G. Two-sided Welch’s t-test or Wald test with Benjamini-Hochberg’s FDR correction method used in B, D, F and H. *p<0.05, **p<0.01, ***p<0.005, or ****p<0.001.

In addition to changes in lipids, carbohydrate metabolism was significantly decreased in HCM as demonstrated by downregulation of GLUT1 (SLC2A1) and SGLT1 (SLC5A1, sodium-glucose linked transporter), reduced levels of glucose, glycolysis intermediates (fructose 6-phosphate, phosphoenolpyruvic acid) and their associated enzymes (HK1, ALDOA, GAPDH, ENO1, PKM), as well as a decrease in pentose phosphate pathway metabolites (ribose-5-phosphate|ribulose 5-phosphate) (Figure 1D, Figure 2B, Figure VA–C in the Supplement). Pyruvate dehydrogenase (PDHA1), which converts pyruvate to acetyl-CoA, a primary link between glycolysis and the TCA cycle, was reduced (Figure 1D, Figure VD in the Supplement). Pyruvate can also enter the TCA cycle by forming malate via malic enzyme 1 (ME1) and pyruvate carboxylase (PC)³², both of which were decreased in HCM (Figure 1D, Figure VD in the Supplement). Notably, TCA cycle intermediates (malate, citrate, succinate) and their associated genes (CS, IDH2, OGDH, SUCLA2, FH, and MDH1) were all decreased (Figure 1D, Figure 2B, Figure 2F). In line with these observations, citrate synthase activity was reduced (Figure 2G). In addition, most amino acids (16/20) as well as key intermediates of nucleotide biosynthesis (NAD, ATP, GDP, CMP) were also decreased, suggesting a global energetic decompensation (Figure 1D, Figure VE–G in the Supplement).

Consistent with the notion of the energetic imbalance in HCM, there was a significant reduction in high energy phosphate molecules (ATP, ADP, PCr) along with reduced expression of genes in the phosphocreatine–creatine kinase (PCr-CK) system including ADP/ATP translocase 1 (SLC25A4), mitochondrial creatine kinase II (CKMT2) and several subunits of ATP synthase machinery (Figure 2B, Figure 2H). These perturbations could compromise energy transfer between mitochondria and the contractile apparatus, exhausting myocardial energy reserve and contributing to the development of contractile dysfunction.

Together these results suggest that HCM pathophysiology induces metabolic alterations that limit fatty acid oxidation and impair entry of several anaplerotic substrates into the TCA cycle. These changes, combined with a decrease in the PCr-CK and ATP synthesis, suggest a globally reduced capacity for mitochondrial oxidative metabolism, ultimately reducing cardiac energy generation in HCM.

HCM is associated with impaired mitochondrial ultrastructure and function

We next sought to characterize the extent to which the above metabolic alterations were associated with alterations in mitochondrial morphology, dynamics and function. Consistent with increased energetic stress, TEM showed a profound disruption in mitochondrial ultrastructure, in which a subset of interfibrillar mitochondria (IFM) were swollen with disorganized and reduced cristae density (Figure 3A). Quantification of cristae density confirmed a marked increase in the percent of mitochondria with severely damaged cristae (Figure 3B–D, Figure VI in the Supplement). To evaluate mitochondrial morphology at a higher resolution, we used electron tomography to reconstruct a 3D volume and found a high variance in cristae density between individual mitochondria in the same sample (Figure 3E, e–e”), consistent with heterogeneous mitochondrial injury. Importantly, these morphological changes were correlated with downregulation of key genes regulating mitochondrial membrane organization, respiratory chain complex assembly, and cristae formation (Figure 3F).

Figure 3. Mitochondrial damage in HCM.

(A) Representative electron micrographs of myocardial tissue from donor and HCM. Each image represents one individual subject (scale bar=2µm). (B-D) Quantitative measurements of interfibrillar mitochondrial cristae density using ImageJ (~20 randomly selected images from each sample). (E) A slice through reconstructed 3D volume and corresponding segmented 3D volumes showing heterogeneous mitochondrial morphology within a single HCM sample (scale bar=100nm). (e-e”) Mitochondrial membranes are color coded in pink and cristae are shown in blue. (F) Chord plot representation of 51 differentially expressed genes (HCM vs control, FDR<0.05) from 7 enriched pathways generated by GOplot. The color map represents fold change of genes (log₂ scale) and p value of go terms (−log₁₀ scale). Error bars represent mean ± SEM. n=3 control and n=14 HCM in B-D; n=7 control and n=13 HCM in F. Between-group comparisons performed using Whitney U test. *p<0.05 or n.s., not significant (p>0.05).

In addition to disruption of cristae architecture, we also found increased mitochondrial number and a trend towards a decrease in mitochondrial size (Figure VII in the Supplement), suggestive of mitochondrial fission, a response to pathologic stress³³. However, expression of key regulators of both mitochondrial fission (Drp1, phosphorylated-Drp1) and fusion (Mfn1/2, Opa1) showed no evidence of activation of these mediators (Figure VIIIA–F in the Supplement). Transcript levels of regulators of mitochondrial biogenesis (PPARGC1, PPARA, NRF1) as well as protein level of PGC1-a were all unchanged (Figure VIIIG and Figure VIIIH in the Supplement). Taken together, these results show that HCM is associated with substantial mitochondrial ultrastructural remodeling, characterized by a reduction in cristae density, but without significant activation of the fission/fusion and biogenesis processes, at least at the clinical time point at which we made this assessment.

We next sought to determine whether these mitochondrial morphologic abnormalities resulted in mitochondrial dysfunction, and thus could contribute to HCM pathogenesis. Consistent with our findings of reduced ATP and ADP content, we found decreased oxidative phosphorylation capacity through complex V (ATP synthase) measured by Oroboros oximeter (Figure 4A, Figure 4B, Figure IXA–C in the Supplement). Activity of complexes II and V and transcript levels of several mitochondrial complex components were also significantly reduced (Figure 4C, Figure IXD in the Supplement). Additionally, UCP2, which uncouples oxygen consumption from ATP synthesis and thus decreases the cellular energy state³⁴, was significantly upregulated (Figure 4D). To ascertain whether reduced ATP levels, in the setting of increased metabolic demand in HCM, activates the AMPK pathway (sensor of low cellular ATP levels³⁵) we measured the catalytic subunit, α-AMPK, and its activated form, pAMPK (Thr172), and found significantly increased pAMPK (Figure 4E–G). Taken together, these data suggest that despite increased metabolic demand and activation of AMPK in HCM hearts, decreased energy supply due to mitochondrial dysfunction contributes to energetic deprivation.

Figure 4. Mitochondrial respiratory function and capacity in HCM.

(A) Average oxygen consumption rate (OCR) showing respiratory activity measured by Oroboros oximeter. Arrows indicate the sequential additions of mitochondrial substrates to assess respiratory states in freshly isolated myocardial tissue. (B) Analysis of ATP production capacity through oxidative phosphorylation (OXPHOS). (C) Mitochondrial complex activities in HCM vs control (spectrophotometry analysis). (D) The transcript level of UCP2 (log₂ scale) measured by RNA-Seq. (E and F) Representative western blot and quantification of phosphorylated- and total AMPK. GAPDH was used as a loading control. (G) ELISA measurements of pAMPK to total AMPK. Error bars represent mean ± SEM. n=3 control and n=9 HCM in A and B; n=5 control and n=5 HCM in C; n=7 control and n=13 HCM in D; n=3 control and n=5 HCM in E and F; n=6 control and n=11 HCM in G. Between-group comparisons performed using Whitney U test in B, C, F, and G. Wald test with Benjamini-Hochberg’s FDR correction method used in D. *p<0.05, ***p<0.005, or n.s, not significant (p>0.05). Mal, malate; Glut, glutamate; Oligo, oligomycin A; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; Anti-A, antimycin A; CI-CV, mitochondrial complex I-V; UCP, uncoupling protein.

Mitochondrial damage is associated with increased Reactive Oxygen Species (ROS)

To investigate mechanisms for HCM mitochondrial structural damage, we investigated the deleterious effects of ROS on mitochondrial lipid membranes and mitochondrial DNA (mtDNA). 4-hydroxynonenal (4-HNE)-modified protein (an end-degradation product of phospholipid peroxidation³⁶) was significantly elevated (Figure 5A, Figure XA in the Supplement). We also found a decrease in various cardiolipin (CL) species including LO3, L2O2, and L3O as well as a trend toward decreased L4, measured by untargeted LC-MS lipidomics (Figure 5B, Figure XB, Figure XC and Excel IV in the Supplement). Cardiolipins are essential for the optimal function of numerous mitochondrial enzymes and are particularly susceptible to ROS-induced oxidation due to their unsaturated fatty acyl composition and proximity to ROS-generating centers in the mitochondria^{37, 38}. Similarly, mtDNA is susceptible to elevated ROS due to the lack of protective histones, inefficient DNA repair mechanisms, and similar proximity to ROS production sites³⁹. Mitochondrial-to-genomic DNA ratios were reduced in HCM (Figure 5C) accompanied by reduced expression of genes associated with mitochondrial DNA integrity and mitochondrial transcription and translation (Figure XD, Figure XE in the Supplement). Interestingly, reduced mtDNA copy number was correlated with decreased cardiolipin species and downregulation of genes associated with cristae formation/maintenance (DNAJ11, TIMM50, SAMM50, C19orf70, TAZ) suggesting that the magnitude of the mtDNA damage coincided with impaired mitochondrial integrity (Figure XF, Figure XG in the Supplement).

Figure 5. Increased oxidative damage in HCM.

(A) 4-hydroxynonenal (4HNE) immunoreactivity indicative of oxidative stress measured by western blot. (B) Major cardiolipin species were measured by mass spec. (C) Quantification of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) using qPCR. (D) Cystine level measured by mass spec (log₂ scale). (E) The ratio of oxidized glutathione (GSSG) to glutathione (GSH) (calculated from mass spec data). (F) The enzymatic activity of glutathione peroxidase (spectrophotometry analysis). (G) Violin plot showing mitophagy associated genes. (H and I) Representative Western blots and quantitative measurements of LC3A and B. GAPDH was used as a loading control. Violin plot was reserved to show RNA-Seq data. Error bars represent mean ± SEM. n=3 control and n=8 HCM in A; n=6 control and n=13 HCM in B, D, and E; n=5 control and n=10 HCM in C; n=5 control and n=5 HCM in F; n=7 control and n=13 HCM in G. n=6 control and n=10 HCM in H and I. Between-group comparisons performed using Whitney U test in A, C, E, F, and I. Two-sided Welch’s t-test and Benjamini-Hochberg’s FDR correction method used in B and D. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 or n.s, not significant (p>0.05). L4, tetralinoleoyl (18:2)₄; L3O, trilinoleoyl-oleoyl (18:2)₃(18:1)₁; L2O2, dilinoleoyl-dioleoyl (18:2)₂(18:1)₂; LO3, linoleoyl-trioleoyl (18:2)₁(18:1)₃; O_.2, superoxide radical; H₂O₂, hydrogen peroxide; GPx, glutathione peroxidase; LC3, Microtubule-associated proteins 1A/1B-light chain 3.

In healthy myocardium, oxidative stress is tightly counterbalanced by reducing agents, coordinated by transcriptional regulation of enzymatic antioxidant systems⁴⁰. Metabolomic data showed high oxidative stress in HCM, indicated by an elevated level of oxidized cysteine (cystine), increased oxidized glutathione (GSSG) to reduced glutathione (GSH) ratio and a reduced level of glutathione, a major antioxidant (Figure 5D, Figure 5E, Figure XH in the Supplement). Levels of other key antioxidants including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSR), and glutathione peroxidase (GPX) were all reduced (Figure XI in the Supplement). Confirming the functional effect of these transcriptional changes, enzymatic activity of glutathione peroxidase (GPx1), which reduces hydrogen peroxide and lipid peroxides⁴¹, was significantly decreased (Figure 5F). These results suggest that increased oxidative stress, due to a combination of elevated ROS and limited antioxidant activity, could contribute to the mitochondrial pathology we observed in HCM.

Impaired mitochondrial quality control/mitophagy

Mitophagy is the principal mechanism for clearing damaged mitochondria and maintenance of myocardial mitochondrial integrity⁴². The abundance of damaged mitochondria in HCM suggests that mitophagic pathways might be either dysfunctionally downregulated or overwhelmed by the degree of mitochondrial damage. We observed a significant downregulation of several genes associated with mitophagy including BNIP3, FUNDC2, ATG9, PINK1, and MAP1LC3 (Figure 5G). LC3 protein expression, which has a central role in autophagosome formation⁴³, was also not increased (Figure 5H, Figure 5I), suggesting a failure to upregulate mitophagy. Similarly, we found no significant difference in Parkin and P62/SQSTM1 expression, which play central roles in autophagic clearance⁴⁴ (Figure XI in the Supplement). Taken together, these data suggest that failure to upregulate or impairment of mitochondrial quality control could be in part responsible for the accumulation of aberrant mitochondria in HCM.

Genotype-phenotype correlation in HCM

Mechanisms by which myocyte-specific mutations, especially those in genes encoding sarcomere proteins, affect the HCM phenotype remain unknown. Additionally, in many HCM patients a disease-causing mutation cannot be identified⁴⁵. To determine the impact of genotype on the phenotypic expression of HCM, we compared key molecular features in patients with or without sarcomeric gene mutations. Unsupervised hierarchical clustering of differentially expressed genes and metabolites revealed no cluster based on genotype (Figure XIIA and Figure XIIB in the Supplement). Levels of FFA, AC (>C14) and carnitine, major altered lipids in HCM, were also similar between the two groups (Figure XIIC–E in the Supplement). Additionally, we found no difference in the percentage of severely damaged mitochondria or oxygen consumption based on patient genotype (Figure XIIF and XIIG in the Supplement). Thus, there were no correlations between the presence of a known mutation and severity of the HCM phenotype, indicating that the HCM metabolomic profile at the time of sampling was relatively homogeneous.

DISCUSSION

Despite several clinical and experimental studies showing metabolic remodeling and inflexibility in hypertrophic and failing hearts^46–48, there are few comprehensive studies in human cardiomyopathies, especially in HCM^{21, 49}. Here, we present an in-depth multi-omics characterization of human HCM myocardium combined with analysis of mitochondrial structure and function. This integrative omics analysis reveals potential molecular mechanisms underlying HCM pathophysiology, including major metabolic derangements across all main biochemical pathways (summarized in Figure 6). These are associated with reduced mitochondrial respiration and accumulation of damaged mitochondria due to a combination of increased oxidative stress, reduced antioxidant defenses, and failure to upregulate mitophagic clearance.

Figure 6. Mitochondrial alterations in HCM.

HCM hearts exhibit impaired fatty acid oxidation and reduced glucose metabolism. Along with these metabolic changes, increased energy demands in hypercontractile HCM hearts enhance ROS production, which together with insufficient antioxidant contents causes damage to diverse mitochondrial sites including mtDNA, cardiolipins, cristae and mitochondrial respiratory complexes. These mitochondrial abnormalities lead to reduced mitochondrial respiration and high energy phosphate molecules ultimately causing myocardial energy deprivation. Italics represents changes at the transcript level while metabolites and enzymatic activities are shown in regular font. Color indicates relative changes in the respective biomolecules: blue, decrease; red, increase. FABP, fatty acid binding protein; CD36, cluster of differentiation 36; CPT1, carnitine palmitoyl transferase type 1; PEP, phosphoenolpyruvate; CoA, Coenzyme A; CI-V, Complex I-V; mtDNA, mitochondrial DNA; TCA, tricarboxylic acid; ACs, acylcarnitines; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, oxidized GSH; SCLC2A1, SLC25A4 and SLC22A5 genes encode glucose transporter1 (GLUT1), the mitochondrial ADP/ATP translocator (ANT1), and carnitine transporter respectively.

Lipids are the preferred substrate for energy production in the normal heart⁵⁰. HCM tissues manifested a substantial accumulation of free fatty acids, whereas the concentration of acylcarnitines and free carnitine were markedly reduced. Our findings are consistent with recent proteomic studies showing downregulation of fatty acid β-oxidation in HCM patients^{21, 45}. Although the underlying mechanism linking these changes to alterations in sarcomere function remains unclear, the increased level of free fatty acids, along with reduced expression of their transporters (SLC27A4, FABP4, and CD36), suggest that the diminished abundance of acylcarnitines is most likely not due to increased fatty acid uptake or retention⁴⁶, but rather to the inability of the heart to increase its capacity to oxidize fatty acids. In support of this hypothesis, we found reduced expression of major enzymes regulating different steps of fatty acid β-oxidation (ACADVL, ACSL1, PGK1, HADHA, ECH1) while the PGC1-PPARA pathway, a major regulator of mitochondrial fatty acid oxidation⁵¹, was not upregulated. Additionally, HCM hearts accumulated toxic lipid intermediates including ceramides. Circulating and myocardial ceramides have been previously shown to be elevated in heart failure and, in some instances, correlated to severity and adverse events^52–54.

In addition to metabolic alterations that limit fatty acid oxidation in HCM, we found reduced levels of glucose and glycolytic metabolites (F6P and PEP) as well as anaplerotic substrates (e.g. aspartate, glutamate, tyrosine, and phenylalanine) that fuel the TCA cycle, ultimately limiting energy provision. Supporting these metabolomic data, the majority of genes catalyzing the TCA cycle (CS, IDH2, OGDH, SUCLA2, FH, and MDH1) were downregulated, leading to reduced levels of malate, citrate, and succinate. These alterations impact downstream energy production through high energy phosphates, with downregulation of key genes involved in mitochondrial energy metabolism including ANT1, CKM2, many subunits of the ETC complex and ATP synthase. These findings are consistent with previous studies suggesting that both HCM and acquired forms of cardiomyopathy are associated with overall reduced cardiac bioenergetics^{10, 11, 15, 18, 45, 55–58}. Imaging assessment of myocardial nucleotide content using phosphorus-NMR (³¹P-NMR) in animal models and humans with HCM^{56, 59–61} detected significant reductions in the PCr/ATP ratio, suggesting limited myocardial energy regenerative capacity. Additionally, both asymptomatic (mutation carriers) and symptomatic HCM patients showed reduced cardiac efficiency using ¹¹C-acetate PET and NMR imaging⁶². Two recent studies^{21, 45} used proteomics analysis to demonstrate that several metabolic pathways related to energy metabolism including oxidative phosphorylation, glycolysis, and β-oxidation were downregulated in HCM. Other evidence supporting an energy deficit in HCM comes from studies of cardiac hypertrophy in inherited syndromes including Barth, Sengers, MELAS and MERRF, in which mitochondrial energy production is impaired^{63, 64}. In addition, patients with mutations in mitochondrial DNA often develop an HCM-like phenotype^{22, 65}. Further evidence supporting the role of altered energetics in HCM is based on mutations of the AMPK gene in families with combined HCM and Wolff-Parkinson-White syndrome^66–68. A small randomized study of HCM patients treated with the metabolic modulator perhexiline, which shifts myocardial substrate utilization from fatty acids to lactate, showed improvements in exercise capacity and New York Heart Association (NYHA) functional class⁶⁹. Thus, impaired energy metabolism could be an important mechanism of functional deterioration in HCM and a potential target for therapeutic intervention.

Increased energy demand in the hypercontractile HCM heart enhances ROS production as by-products of oxidative phosphorylation⁴. Elevated ROS and insufficient antioxidant protection expose mitochondria to increased oxidative stress leading to mitochondrial damage⁷⁰. ROS react with DNA, proteins and lipids (especially mitochondrial membranes), inactivating ETC complexes and mitochondrial proteins and impairing mitochondrial respiration^{21, 71}. We found increased oxidative stress markers, including 4HNE, GSSG, and cystine in HCM hearts, while transcript levels of several major antioxidants (GPX1, GSR, SOD, CAT) and enzymatic activity of GPx were significantly diminished. HCM myocardium demonstrates a reduction in several cardiolipin species, shown to impair mitochondrial respiratory activity by destabilizing inner mitochondrial membrane integrity, feed-forwarding additional electron leakage, and further increasing ROS formation⁷². Similar to previous studies in hypertrophied and failing hearts^73–75, mtDNA was reduced in HCM, along with downregulation of mitochondrial genes encoding ribosomal, tRNA, and ETC subunits. Finally, our systematic analysis of mitochondrial morphology using quantitative electron microscopy revealed a significant subset of severely damaged mitochondria with reduced cristae density. Cristae loss may be explained by the reduced expression of several genes associated with cristae formation and maintenance of cristae junctions. CHCHD3, a member of cristae organizing proteins (MICOS) and genes encoding DNAJC11 and SAMM50 which interact with the MICOS complex⁷⁶ for proper cristae formation⁷⁷ were all significantly downregulated.

Mitochondrial structural alterations seen in HCM were associated with reduced mitochondrial oxidative phosphorylation capacity and decreased respiratory complex activity, confirming mitochondrial functional impairment. Previous studies have reported mitochondrial dysfunction and abnormal morphology in HCM^{19, 20, 27}. In pigs, there were signs of swollen mitochondria with disrupted cristae, mtDNA depletion, and reduced mitochondrial complex activity⁷⁸. In a single case study, an end-stage heart failure patient with HCM who underwent heart transplant showed lowered respiratory capacity in mitochondria isolated from the septum compared to other regions of the heart⁷⁹. Interestingly, patients with end-stage heart failure who underwent left ventricular assist device placement showed an overall increase in substrate oxidation and improved mitochondrial function after mechanical unloading, suggesting reversibility of these defects⁸⁰. Consistent with this result, we found broad heterogeneity in the degree of cristae damage within the same patient sample, suggesting that a subset of mitochondria in HCM myocardium may still have normal respiratory function but may be insufficient to meet the high energy demands of the hypercontractile HCM heart.

Activation of the mitophagy pathway to eliminate aberrant mitochondria with reduced respiratory capacity helps to prevent the cytotoxic impact of ROS, maintaining cellular homeostasis and improving mitochondrial function^{11, 81–84}. Despite the presence of damaged mitochondria, we found downregulation of several genes associated with mitophagy including BNIP3, FUNDC2, ATG9, PINK1, and MAP1LC3. LC3 protein expression was also not changed, suggesting a failure to upregulate mitochondrial quality control pathways in the presence of significant mitochondrial damage. Similar to our results, autophagy was found to be defective in mice-carrying MYBPC3 mutations and in a small group of HCM patients⁸⁵. Activation of autophagy, was shown to attenuate cardiomyopathy in these mice⁸⁵. We speculate that the presence of so many aberrant mitochondria in HCM could be due to failure of mitophagic mitochondrial clearance. Although accumulation of autophagic vacuoles caused by defective proteolytic systems has also been associated with HCM pathology^{82, 86}, the lack of LC3 upregulation combined with the absence of accumulated autophagic vesicles in our EM data suggest that autophagy is not activated, at least at this stage of the disease. Altogether, impaired mitochondrial quality control is likely to contribute to the accumulation of damaged mitochondria in HCM, ultimately leading to progressive mitochondrial damage, a vicious cycle in the progression of the HCM phenotype.

A recent study by Coats et al.²¹, using label-free proteomics to characterize HCM tissue, corroborates some of our findings, including alterations in proteins associated with energy metabolism and mitochondrial dysfunction. In line with our data, they showed reduced β-oxidation and downregulation of ACSL1, ACADVL, and HADHB. Similar to our data, there were decreased levels of mitochondrial respiratory complex proteins despite no change in citrate synthase, implying similar mitochondrial number. Coates et al. hypothesized that mtDNA depletion or perturbation in HCM could result in alteration of the electron transport chain. In support of their hypothesis, we found reduced mtDNA, previously reported in pigs with HCM⁷⁸ as well as in patients with hypertrophy due to congenital heart disease⁷⁴. Although we did not measure the protein expression of citrate synthase, its enzymatic activity was reduced in HCM, confirming limited energy metabolism. Besides validation of some of our data by Coats et al., our integrated omics approach provides a more in-depth analysis of metabolic changes in HCM. Importantly, by combining omics data with mitochondrial morphological and functional assessments our study provides mechanistic insights into cellular processes such as the role of oxidative stress and reduced antioxidant defenses on the observed metabolic alterations and reduced metabolic reserve. We believe our approach of using combinational analysis from different modalities is a robust way to understand the multiple perturbations in a disease as complex as HCM.

Overall, our findings suggest that altered metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms among HCM patients undergoing myectomy surgery, a time when their global cardiac function is still hyperdynamic, and far from end-stage heart failure. Using an unbiased approach, we observed similar molecular changes in the HCM cohort independent of whether a sarcomeric gene mutation was present or not. Consistent with this finding, two recent studies^{45, 87} showed similar proteomic and proteoform profiles in HCM patients regardless of their individual gene mutation, suggesting that secondary mechanisms (e.g. cellular and mitochondrial stress) lead, through common pathways, to the HCM clinical phenotype. HCM mutations in sarcomeric proteins primarily cause diverse alterations in biophysical properties of the cardiomyocyte, which ultimately perpetuate the hypertrophic response in the heart. The molecular and histologic alterations (e.g. alterations in cardiac energetics) that occur in response to these primary changes are influenced by a combination of genetic, epigenetic, and environmental factors⁸⁸. Therefore, whereas the initial alterations are divergent, they converge into common pathways that lead to altered metabolism, which we have identified as a major component of the HCM phenotype⁸⁸.

LIMITATIONS

This study represents the most comprehensive multi-omics and functional characterization of HCM in human myocardium. However, there are several limitations associated with a study of this kind. First, the samples were obtained from HCM patients undergoing surgical myectomy, only providing us with a narrow temporal window into the disease. Thus, we cannot extrapolate our findings to earlier or later stages of the HCM disease process. All of our patients were undergoing myectomy for symptomatic LVOT obstruction, so they represent a sub-class (hypertrophic obstructive cardiomyopathy), albeit a large one, of all HCM patients. Importantly, the vast majority of our patients had hyperdynamic left ventricular function, and the remainder had preserved function, thus these samples were from hearts that had not yet developed many of the secondary and tertiary changes associated with the transition from hypercontractility to heart failure. It is therefore even more notable that we found such a high degree of metabolic and mitochondrial alterations in patients who were at this stage in their disease. Second, global metabolomic and transcriptomic studies of human heart tissue, especially in HCM, are influenced by multiple factors in addition to disease stage, including heterogeneity within the myocardium (septum, apex, free wall), genetic diversity between patients, secondary remodeling mechanisms, medical comorbidities, and drug therapy. It is possible that the septum represents a more advanced stage of HCM pathophysiology compared to the LV free wall in these patients. Third, metabolic measurements preclude definitive conclusions on metabolic flux changes. However, when combined with expected changes in expression of their regulatory enzymes, provide insights into likely mechanisms explaining the metabolic alterations. Future tracing experiments with isotopically-labeled metabolites are warranted to confirm metabolic flux dysregulation in HCM. Nevertheless, our extensive metabolic profiling provides a global map of altered metabolic pathways. Fourth, our sample size limited our ability to perform correlation analysis between any individual sarcomeric mutation and HCM phenotype, warranting further exploration in a larger cohort. Fifth, most of the control samples were obtained from unused donor hearts, and thus were from individuals who suffered catastrophic neurological injury, suggesting the potential for metabolic alterations in the controls. However, all donor hearts had normal function at the time of harvesting. Both control and HCM hearts were subjected to cardioplegia, although the duration was longer for the donor controls; importantly none were longer than 4 hours. Finally, we cannot eliminate the effect of regional heterogeneity on our analysis. Different regions of the left ventricle were assessed in our groups as follow: LV septal tissue in HCM, interventricular septum and LV apex in donors, and LV free wall in mitral stenosis patients.

CONCLUSION

In summary, our study demonstrates the power of using an unbiased integrated multi-omic analysis to determine the role of global alterations in cardiac metabolism and energetics as a pathophysiologic mechanism in HCM. The correlation of diffuse metabolomic and lipidomic alterations with changes in transcription of key enzymes regulating those pathways strengthens this evidence and provides mechanistic insights. Accompanying these metabolic derangements, we found increased oxidative damage leading to dramatic mitochondrial structural and functional defects, exacerbated by a failure to upregulate mitochondrial quality control, and thereby increasing energetic compromise. The degree to which these alterations are present at this earlier stage suggests that developing new strategies directed toward improving metabolic and mitochondrial function could be promising approaches to slow down the progression of HCM.

Clinical Perspective.

What is new?

• We leveraged a novel integrated multi-omics platform to explore the role of altered cardiac metabolism as a common pathway leading to the pathophysiological phenotypes in patients with hypertrophic cardiomyopathy (HCM).

• We describe widespread alterations across a broad array of metabolic pathways and in mitochondrial structure and function at a relatively early stage in the progression of HCM.

• These findings provide a crucial connection between two central aspects of HCM disease: biophysical properties of the cardiomyocyte and cellular metabolism.

What are the clinical implications?

• This degree of metabolic derangement has not been previously suspected in patients with HCM, especially at this stage of disease, when contractile function is still hyperdynamic.

• Our integrated multi-omics approach should be applicable to diverse forms of cardiovascular disease for discovery of both mechanism and drug targets.

• These findings highlight the potential for development of new HCM therapeutics targeting metabolic alterations, in addition to therapies targeting the sarcomere.

Non-standard Abbreviations and Acronyms

HCM

Hypertrophic Cardiomyopathy

ROS

Reactive Oxygen Species

LV

Left Ventricle

LVOT

LV Outflow Tract

TEM

Transmission Electron Microscopy

FFA

Free Fatty Acid

AC

Acylcarnitine

CER

Ceramide

TAG

Triacylglycerol

DAG

Diacylglycerol

PCr

Phosphocreatine

PCr-CK

Phosphocreatine–Creatine Kinase