Differences in molecular phenotype in mouse and human hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is characterized by phenotypic heterogeneity. We investigated the molecular basis of the cardiac phenotype in two mouse models at established disease stage (mouse-HCM), and human myectomy tissue (human-HCM). We analyzed the transcriptome in 2 mouse models with non-obstructive HCM (R403Q-MyHC, R92W-TnT)/littermate-control hearts at 24 weeks of age, and in myectomy tissue of patients with obstructive HCM/control hearts (GSE36961, GSE36946). Additionally, we examined myocyte redox, cardiac mitochondrial DNA copy number (mtDNA-CN), mt-respiration, mt-ROS generation/scavenging and mt-Ca²⁺ handling in mice. We identified distinct allele-specific gene expression in mouse-HCM, and marked differences between mouse-HCM and human-HCM. Only two genes (CASQ1, GPT1) were similarly dysregulated in both mutant mice and human-HCM. No signaling pathway or transcription factor was predicted to be similarly dysregulated (by Ingenuity Pathway Analysis) in both mutant mice and human-HCM. Losartan was a predicted therapy only in TnT-mutant mice. KEGG pathway analysis revealed enrichment for several metabolic pathways, but only pyruvate metabolism was enriched in both mutant mice and human-HCM. Both mutant mouse myocytes demonstrated evidence of an oxidized redox environment. Mitochondrial complex I RCR was lower in both mutant mice compared to controls. MyHC-mutant mice had similar mtDNA-CN and mt-Ca²⁺ handling, but TnT-mutant mice exhibited lower mtDNA-CN and impaired mt-Ca²⁺ handling, compared to littermate-controls. Molecular profiling reveals differences in gene expression, transcriptional regulation, intracellular signaling and mt-number/function in 2 mouse models at established disease stage. Further studies are needed to confirm differences in gene expression between mouse and human-HCM, and to examine whether cardiac phenotype, genotype and/or species differences underlie the divergence in molecular profiles.

Figure 1

Mouse Phenotyping (a) Study design. (b) Echocardiography: Representative 2D and M-mode images from mutants (MyHC, TnT) and respective littermate control mice (ContM, ContT) illustrate that Left Ventricular (LV) cavity size is smaller in TnT-mutant mice, and larger in MyHC-mutant mice, when compared to littermate-controls. (c) Representative gross anatomy (left panel) and histology images (right panel) from mutants (MyHC, TnT) and littermate controls (ContM, ContT) reveal larger heart size in MyHC-mutants, and smaller heart size in TnT-mutants, when compared to littermate-controls. Bi-atrial enlargement is prominent in TnT-mutant mice. (d) Picrosirius Red staining reveals higher interstitial fibrosis in both mutant mouse hearts when compared to littermate-control hearts (for gross anatomy, calibration markers are 1 mm; microscopy calibration marker indicates 50 μm). For complete list of data with statistical analysis please see Table 1.

Figure 2

Global miRNA-seq analysis of MyHC-mutant, TnT-mutant mouse hearts and human-HCM. (a,b) Volcano plots of normalized miRNA reads in hearts of (a) MyHC-mutant mouse hearts, (b) TnT-mutant mouse hearts and littermate-control hearts (ContM and ContT). Each dot represents individual miRNA. The values are means of three biological replicates. Significantly (adjusted p < 0.05) upregulated miRNAs (miR-99b-5p, miR-195a-5p, miR-497a-5p) are shown in magenta and significantly downregulated miRNA (miR-150-5p) is shown in cyan in (b). miRNAs with > 300 normalized reads in any of the genotypes are shown. (c,d) Volcano plot of log2(fold-change) of miRNA-seq results in (c) MyHC-mutants and (d) TnT-mutants compared to littermate-controls. Benjamini–Hochberg method was used to adjust p values for multiple comparisons (n = 3 biological replicates). (e,f) Heatmap of log2(fold-change) of normalized miRNA reads in MyHC-mutants and TnT-mutants compared with littermate-controls (means of three biological replicates are presented). miRNAs are sorted in descending order of fold-change in abundance in (e) MyHC-mutants and (f) TnT-mutants. (g) Normalized miRNA abundance of miR-150-5p, miR-99b-5p, miR-195a-5p, and miR-497a-5p (mean ± S.D. n = 3 biological replicates; *p < 0.05 using 2-sided unpaired Student’s t-test). (h) Volcano plots of normalized miRNA reads in human myectomy tissue and control-hearts. (i) Heatmap of log2(fold-change) of normalized miRNA reads in human myectomy tissue (n = 107) compared to control-hearts (n = 20). miRNAs are ordered in descending order of fold-change in abundance (means are presented). (j) Normalized miRNA abundance of miR-150-5p, miR-99b-5p and miR-195a-5p (mean ± S.D.; n = 107 HCM patients/n = 20 controls; *p < 0.05 using 2-sided unpaired Student’s t-test).

Figure 3

Global mRNA-seq analysis of MyHC and TnT-mutant mouse hearts and human-HCM. (a) Volcano plots of mRNA expression. X-axis shows mean of normalized counts among the 6 biological samples tested (3 biological replicates each for two genotypes compared in each graph) and Y-axis shows log2(fold-change) in the normalized counts. (b) Venn diagram of the numbers of differentially expressed genes. (c) Heatmaps of log2(fold-change) of mRNA expression levels in MyHC-mutant and TnT-mutant mouse hearts compared with littermate-control hearts for the 150 and 186 genes that are differentially expressed (adjusted p < 0.05) in either MyHC or TnT-mutant mice, respectively (means of three biological replicates are presented). The genes are sorted based on log2(fold-change) in MyHC-mutant mice (left panel) or in TnT-mutant mice (right panel). (d) Heatmap of log2(fold-change) of top 20 upregulated genes and downregulated genes in MyHC-mutant mice compared to littermate-controls (means of three biological replicates are presented). (e) Heatmap of log2(fold-change) of top 20 upregulated and downregulated genes in TnT-mutant mice compared to littermate-controls (means of three biological replicates are presented). (f) Heatmap of log2(fold-change) of top 20 upregulated and downregulated genes in human myectomy tissue compared to control hearts (means data is presented; n = 105 HCM patients/n = 39 controls).

Figure 4

Differential metabolic gene expression in MyHC and TnT-mutant mouse hearts and human-HCM. (a) Heatmap of log2(fold-change) of normalized mRNA reads in MyHC-mutant and TnT-mutant mouse hearts compared with littermate-control hearts. mRNAs are clustered depending on the metabolic function of the involved metabolic genes (Krebs cycle, Fatty Acid metabolism, Glucose use, BCAA, OxPhos). Means of three biological replicates are presented. (b,c,e) Volcano plots of normalized mRNA reads of metabolic genes in MyHC-mutant and TnT-mutant mice compared to littermate-controls and in human myectomy tissue and control-hearts. Benjamini–Hochberg method was used to adjust p values for multiple comparisons; n = 3 biological replicates. (d) Heatmap of log2(fold-change) of normalized mRNA reads in human myectomy tissue compared to control-hearts (means are presented; n = 105 HCM patients/n = 39 controls). mRNAs are clustered depending on the metabolic function of the involved metabolic genes (Krebs cycle, Fatty Acids, Glucose use, BCAA, OxPhos).

Figure 5

Myocyte and mitochondrial studies in mutant and littermate control mouse hearts. (a) Mitochondrial DNA copy number (mtDNA-CN) is presented as relative copy number of COX/GAPDH. MtDNA-CN in mutants was normalized to littermate-control data. MyHC-mutants had similar mtDNA-CN, whereas TnT-mutants had lower mtDNA-CN when compared to littermate-controls (n = 9 hearts in each group; *p = 0.02 using 2-sided unpaired student’s t-test). (b–d) Two-photon microscopy: (b) Representative images of cardiac myocytes. From right to left, unstained myocyte (transmitted light) and stained with MCB (blue) and TMRM (red) to simultaneously monitor reduced glutathione (GSH) and mitochondrial membrane potential (Δψ_m) respectively. (c) Both mutant mouse myocytes had lower levels of GSH when compared to controls. ΔΨ_m was more hyperpolarized in MyHC-mutants, but similar in TnT-mutants, when compared to littermate-controls. (d) NAD(P)H levels were similar, but NAD(P)H pool levels were lower in both mutants when compared to littermate-controls (n = 3 mice in each group with ≥ 30 cells imaged per animal; *p < 0.05, **p < 0.01 using 2-sided unpaired student’s t-test. Fluorescence unit (FU) data is normalized to littermate-controls. %NAD(P)H was calculated based on NAD(P)H FU, normalized to respective NAD(P)H pool). (e–g) Mitochondrial respiration: (e) Complex I respiration: MyHC-mutant mitochondria had higher state 4 respiration, whereas TnT-mutants had lower state 3 respiration resulting in lower complex I respiratory control ratio (RCR) in both mutants when compared to littermate-controls. (f) Complex II respiration: Only TnT-mutants had lower state 3 respiration compared to littermate-controls. (g) Complex IV respiration: Only TnT-mutants exhibited lower complex IV respiration when compared to littermate-controls (n = 8 mice in ContM and TnT-mutants; n = 10 mice in ContT; n = 12 mice in MyHC-mutants; *p < 0.05, using 2-sided unpaired student’s t-test). (h–j) Mitochondrial H₂O₂ (ROS) generation: MyHC-mutant mitochondria demonstrated lower ROS during state 4 with glutamate/malate but similar ROS during State 3 as littermate-controls. MyHC-mutants showed similar ROS with succinate ± rotenone. TnT-mutants demonstrated lower ROS emission in state 3 with succinate ± rotenone (n = 8 mice for ContT and TnT-mutants; n = 10 mice for ContM and MyHC-mutants; *p < 0.05, using 2-sided one sample t-test). (k) Mitochondrial NAD(P)H: MyHC-mutant mitochondria had lower levels of reduced NAD(P)H during state 4, but higher levels during state 3 compared to littermate-controls; TnT-mutants had similar reduced NAD(P)H in state 3/4 as littermate-controls (n = 10 mice for ContT; n = 12 mice for ContM and TnT-mutants; n = 14 mice for MyHC-mutants. *p < 0.05, **p < 0.01 using 2-sided unpaired student’s t-test). (l) Mitochondrial Ca²⁺ handling: Mitochondria were pre-incubated with Fura-FF (20 μΜ) to monitor [Ca²⁺] changes in the mitochondrial matrix. Calcium Green-5N (0.1 μM) was added at the beginning of the experiment to monitor extra-mitochondrial [Ca²⁺] changes. Mitochondria were energized with glutamate/malate (GM) and additions of CaCl₂ followed until the opening of PTP occurred (reflected by abrupt slow increase in the extra-mitochondrial calcium signal and slow decrease in the intra-mitochondrial calcium signal. Traces of extramitochondrial (l1, l3) and intramitochondrial (l2, l4) calcium signal in mutant TnT or MyHC (red trace) and littermate control (black trace) mitochondria. l5. Bar graphs summarizing mitochondrial Ca²⁺ handling Mitochondrial permeability transition pore (mPTP) activation occurred at similar [Ca²⁺] as littermate-controls in MyHC-mutants, but at lower [Ca²⁺] than littermate-controls in TnT-mutants. When compared to littermate-controls, matrix [Ca²⁺]_free was similar in MyHC-mutants and lower in TnT-mutants (n = 10 mice for MyHC-mutants/controls; n = 8 mice for TnT-mutants/controls; *p < 0.05, using 2-sided one sample t-test). *(a–l): all comparisons were made for each group (ContM vs MyHC and ContT vs TnT). (m) Redox-optimized ROS balance: Both mutant mouse hearts are characterized by a lower-energy oxidized redox environment, due to lower reduced GSH and NAD(P)H pool levels, despite lack of increase in mitochondrial ROS generation.

Figure 5

Myocyte and mitochondrial studies in mutant and littermate control mouse hearts. (a) Mitochondrial DNA copy number (mtDNA-CN) is presented as relative copy number of COX/GAPDH. MtDNA-CN in mutants was normalized to littermate-control data. MyHC-mutants had similar mtDNA-CN, whereas TnT-mutants had lower mtDNA-CN when compared to littermate-controls (n = 9 hearts in each group; *p = 0.02 using 2-sided unpaired student’s t-test). (b–d) Two-photon microscopy: (b) Representative images of cardiac myocytes. From right to left, unstained myocyte (transmitted light) and stained with MCB (blue) and TMRM (red) to simultaneously monitor reduced glutathione (GSH) and mitochondrial membrane potential (Δψ_m) respectively. (c) Both mutant mouse myocytes had lower levels of GSH when compared to controls. ΔΨ_m was more hyperpolarized in MyHC-mutants, but similar in TnT-mutants, when compared to littermate-controls. (d) NAD(P)H levels were similar, but NAD(P)H pool levels were lower in both mutants when compared to littermate-controls (n = 3 mice in each group with ≥ 30 cells imaged per animal; *p < 0.05, **p < 0.01 using 2-sided unpaired student’s t-test. Fluorescence unit (FU) data is normalized to littermate-controls. %NAD(P)H was calculated based on NAD(P)H FU, normalized to respective NAD(P)H pool). (e–g) Mitochondrial respiration: (e) Complex I respiration: MyHC-mutant mitochondria had higher state 4 respiration, whereas TnT-mutants had lower state 3 respiration resulting in lower complex I respiratory control ratio (RCR) in both mutants when compared to littermate-controls. (f) Complex II respiration: Only TnT-mutants had lower state 3 respiration compared to littermate-controls. (g) Complex IV respiration: Only TnT-mutants exhibited lower complex IV respiration when compared to littermate-controls (n = 8 mice in ContM and TnT-mutants; n = 10 mice in ContT; n = 12 mice in MyHC-mutants; *p < 0.05, using 2-sided unpaired student’s t-test). (h–j) Mitochondrial H₂O₂ (ROS) generation: MyHC-mutant mitochondria demonstrated lower ROS during state 4 with glutamate/malate but similar ROS during State 3 as littermate-controls. MyHC-mutants showed similar ROS with succinate ± rotenone. TnT-mutants demonstrated lower ROS emission in state 3 with succinate ± rotenone (n = 8 mice for ContT and TnT-mutants; n = 10 mice for ContM and MyHC-mutants; *p < 0.05, using 2-sided one sample t-test). (k) Mitochondrial NAD(P)H: MyHC-mutant mitochondria had lower levels of reduced NAD(P)H during state 4, but higher levels during state 3 compared to littermate-controls; TnT-mutants had similar reduced NAD(P)H in state 3/4 as littermate-controls (n = 10 mice for ContT; n = 12 mice for ContM and TnT-mutants; n = 14 mice for MyHC-mutants. *p < 0.05, **p < 0.01 using 2-sided unpaired student’s t-test). (l) Mitochondrial Ca²⁺ handling: Mitochondria were pre-incubated with Fura-FF (20 μΜ) to monitor [Ca²⁺] changes in the mitochondrial matrix. Calcium Green-5N (0.1 μM) was added at the beginning of the experiment to monitor extra-mitochondrial [Ca²⁺] changes. Mitochondria were energized with glutamate/malate (GM) and additions of CaCl₂ followed until the opening of PTP occurred (reflected by abrupt slow increase in the extra-mitochondrial calcium signal and slow decrease in the intra-mitochondrial calcium signal. Traces of extramitochondrial (l1, l3) and intramitochondrial (l2, l4) calcium signal in mutant TnT or MyHC (red trace) and littermate control (black trace) mitochondria. l5. Bar graphs summarizing mitochondrial Ca²⁺ handling Mitochondrial permeability transition pore (mPTP) activation occurred at similar [Ca²⁺] as littermate-controls in MyHC-mutants, but at lower [Ca²⁺] than littermate-controls in TnT-mutants. When compared to littermate-controls, matrix [Ca²⁺]_free was similar in MyHC-mutants and lower in TnT-mutants (n = 10 mice for MyHC-mutants/controls; n = 8 mice for TnT-mutants/controls; *p < 0.05, using 2-sided one sample t-test). *(a–l): all comparisons were made for each group (ContM vs MyHC and ContT vs TnT). (m) Redox-optimized ROS balance: Both mutant mouse hearts are characterized by a lower-energy oxidized redox environment, due to lower reduced GSH and NAD(P)H pool levels, despite lack of increase in mitochondrial ROS generation.

Figure 6

Ingenuity pathway analysis: top 5 dysregulated signaling pathways (a), transcriptional factors (b) and therapies (c) are presented for MyHC-mutant (1) TnT-mutant (2) mouse hearts, and human myectomy tissue (3). All comparisons are made to corresponding controls. Cutoff value for significance of p < 0.01 and |Z-score|> 1. Comparison of mRNA data from mutant HCM mice and human myectomy samples with respective controls was performed by ANOVA using the Partek Genomics Suite 7.0 platform.

Figure 7

Genes implicated in cardiac hypertrophy and its regulation: volcano plots of normalized mRNA reads in MyHC and TnT-mutant mouse hearts (a,b) and human myectomy tissue (c). Heatmap of log2(fold-change) of differentially expressed genes related to hypertrophy and its regulation in MyHC-mutant and TnT-mutant mouse hearts (d) and in human myectomy tissue (e) compared to respective controls. Benjamini–Hochberg method was used to adjust p values for multiple comparisons; n = 3 biological replicates for mouse data; n = 105 HCM patients/n = 39 controls for human data). Genes involved in TGF-β signaling pathway and its regulation: Volcano plots of normalized mRNA reads of TGF-β pathway related genes in MyHC-mutant and TnT-mutant mouse hearts (a,b) and human myectomy tissue (c). Heatmap of log2(fold-change) of differentially expressed genes related to TGF-β signaling pathway and its regulators in MyHC-mutant and TnT-mutant mouse hearts (d) and in human myectomy tissue (e) compared to respective controls. Benjamini–Hochberg method was used to adjust p values for multiple comparisons (n = 3 biological replicates for mouse data; n = 105 HCM patients/n = 39 controls for human data).

Figure 8

Genes involved in TGF-β signaling pathway and its regulation: Volcano plots of normalized mRNA reads of TGF-β pathway related genes in MyHC-mutant and TnT-mutant mouse hearts (a,b) and human myectomy tissue (c). Heatmap of log2(fold-change) of differentially expressed genes related to TGF-β signaling pathway and its regulators in MyHC-mutant and TnT-mutant mouse hearts (d) and in human myectomy tissue (e) compared to respective controls. Benjamini–Hochberg method was used to adjust p values for multiple comparisons (n = 3 biological replicates for mouse data; n = 105 HCM patients/n = 39 controls for human data).

Figure 9

Hypothesis schematic: HCM mutations lead to abnormalities in Ca²⁺ handling, oxidative stress, energetic stress which lead to cardiac transcriptional changes, mitochondrial dysfunction and metabolic gene remodeling. Mitochondrial dysfunction and altered cardiac metabolism contribute to cardiac hypertrophy/fibrosis and exacerbate calcium mishandling, oxidative/energetic stress.