 interaction networks (Figure 4). The first, most extensive network includes the growth factors, EGF and VEGF, as important nodes that are reduced in the postoperative state and linked to reduction of pro-inflammatory cytokines and signaling molecules related to inflammation, including IL25, TNSF12, TNFSF14, TNFRSF12A, TNFRSF18, and IL10RB (Figure 4A). Interestingly, many intracellular signaling molecules are present as network nodes but not present in the actual proteomic dataset, such as ERK1, SRC, RAS, and tyrosine kinase. Lack of signal for these nodes is not surprising since the proteomic dataset is from proteins circulating in plasma and they are either not detected or not represented in the SOMAscan assay. Their presence in the network suggests important links between circulating extracellular proteins and intracellular signaling pathways. A second network includes MMP12 as a central node with increased expression in the postoperative state, accompanied by increased expression of additional extracellular matrix (ECM) proteases, ADAMTS15, and elastase, as well as the ECM protein periostin (POSTN) and the intercalated disc protein, CDON (Figure 4B). This network is linked to intracellular AKT signaling and various ECM proteins. A third network is focused on VEGFA, Ap1, LDL, and GSTP1, and is linked to intracellular NOS and NFκB signaling (Figure 4C).
Modeling the links between myectomy-associated proteins and shared upstream regulatory proteins was particularly informative. In Figure 5A, the upstream regulators that are most significantly enriched, based on p-value overlap, by the input of the 79 proteins are shown. The entire list of predicted upstream regulators, with their associated p-values and z-scores is shown in Supplementary Table S1. Analysis of the predicted positive upstream regulators for the differentially expressed proteins converged on Tumor Necrosis Factor (TNF), Interferon γ (IFNγ), and Transforming Growth Factor β1 (TGFβ1), as most significant. Thirty out of 79 proteins are predicted downstream targets of the pro-inflammatory cytokine TNF (Figure 5B), while 24 proteins are downstream of IFNγ (Figure 5C) and 28 proteins are downstream of TGFβ1 (Figure 5D). Several proteins mentioned above as being linked to cardiovascular disease, cardiomyopathy, or vascular disease are highlighted by the red arrows in the upstream regulator networks. The proteins that increased after surgery are denoted by the red symbols, and the ones that decreased are denoted by green symbols. These results indicate that these signaling nodes are likely involved in the dysregulation of a sizeable portion of the top 79 proteins in the myectomy signature. Predicted negative upstream regulators of interest include Epidermal Growth Factor (EGF), FOS, and CD28, shown with their downstream targets (Figure 6A–C). An additional predicted positive upstream regulator, CSF1, is also shown with its downstream targets (Figure 6D).
In Figure 7, a selected set of the most significantly enriched biological functions, based on p-value overlap, using the 79 proteins as input (p < 0.05) are shown. Among the significantly affected functional categories, enrichment for biological functions linked to “Inflammation of Organ” were most prominent, followed by biological processes associated with leukocyte migration, cell movement of leukocytes, fibrosis, vasculogenesis, angiogenesis, development of vasculature, chronic inflammatory disorder, and cell movement of mononuclear leukocytes (Figure 7). The entire list of predicted biological functioons with their associated p-values and z-scores is shown in Supplementary Table S2. Other highly enriched key biological functions of particular interest with regard to cardiomyopathy include acute coronary event, myocardial infarction, acute myocardial infarction, abnormal morphology of cardiovascular system, and morphology of cardiovascular system (Figure 7). Metabolism of eicosanoid and synthesis of eicosanoid further support the notion of inflammatory processes contributing to the surgery effect (Figure 7). Figure 8 highlights in detail the 23 proteins linked to vascular functions among the 79 proteins (Figure 8A), the 19 proteins associated with fibrosis (Figure 8B), the 11 proteins linked to cardiovascular infarction (Figure 8C), and the 14 proteins linked to eicosanoid metabolism, synthesis, and release (Figure 8D).
We found that plasma protein profiles from HCM patients with LVOT obstruction can distinguish the preoperative from the postoperative state, and surgical myectomy results in a reduction of circulating plasma proteins, associated with a proinflammatory state. The association between HCM and a proinflammatory state was consistent with previous reports [4,14], but our study was the first, to the best of our knowledge, to demonstrate a potential improvement after surgical myectomy. The potential mechanisms through which HCM leads to systemic inflammation are not clear, but are associated with myocardial fibrosis, which might be secondary to cardiomyocyte injury. A possible mechanism through which surgery to relieve outflow tract obstruction alleviates myocardial injury might involve reduction of subendocardial ischemia from elevated LV filling pressures. In this context, it is interesting to see the reduction in nicotinamide phosphoribosyl-transferase (NAMPT) after surgery. This enzyme plays a significant role in cerebral ischemia [15], hypertension, atherosclerosis, heart failure [10], and ischemic heart disease [16]. Additionally, inflammatory processes and NAMPT inhibitors were shown to protect against neuronal injury in animal models (reviewed in [11]).
Another interesting finding was that circulating matrix associated proteases, such as MMP12, show increased levels, along with circulating matrix proteins, in the postoperative state. One possible explanation is that the increase in matrix remodeling enzymes is a consequence of wound healing and the postoperative state, although one would expect that surgical wound healing would be completed by the three month follow-up visit. A more intriguing possibility is that surgical relief of LVOT obstruction is associated with prolonged extracellular matrix remodeling in the HCM heart that retards the development of interstitial fibrosis seen in advanced cases.
A potential role for angiogenesis in the pathogenesis of HCM was not previously established. A role for angiogenesis in cardiac hypertrophy, however, was demonstrated repeatedly in experimental models (reviewed in [17]). Increased capillary vascularity is thought to support the increased circulatory and metabolic demands of the hypertrophied cardiomyocyte. Increased angiogenesis in HCM might also occur in response to subendocardial ischemia. A reduction in circulating proangiogenic factors after myectomy, as suggest by the data in this study, is an unexpected finding, but again might reflect a reduction in subendocardial ischemia, after reduction of elevated filling pressures.
Our work also identified potential upstream regulators of systemic inflammation and fibrosis, such as TNFα, IFNγ, and TGFβ1. EGF, another identified upstream regulator, is also known to promote angiogenesis through induction of autocrine VEGF expression [18], to regulate inflammation, through its effects on TNSRSF12A (also known as FN14) [19] and to regulate matrix turnover through effects on MMP12, which itself regulates angiogenesis and inflammation [20]. The cellular oncogene c-fos, also found in our screen for upstream regulators, promotes angiogenesis through the induction of VEGF [21]. CSF1, another upstream regulator identified in our analysis, controls the production, differentiation, and function of macrophages [22], and thus is also an important regulator of inflammation. CD28, another upstream regulator, is involved in T-cell activation, induction of cell proliferation and cytokine production and promotion of T-cell survival, and thus is also involved in regulation of the immune response [23]. These upstream regulators might provide potential therapeutic targets in obstructive HCM.
A recent study examined plasma proteomic profiles in patients with aortic stenosis, before and after transcatheter valve replacement [24]. Aortic stenosis results in fixed outflow tract obstruction from abnormal narrowing of the aortic valve, and transcatheter aortic valve replacement relieves the obstruction. In HCM, LVOT obstruction is dynamic, and to date a comparison of plasma profiles in dynamic vs. fixed obstruction is not done. The published aortic stenosis dataset using the same SOMAscan technology implicates MAP kinase signaling, HIPPO signaling, and focal adhesion pathways, as important mediators of myofibroblast activation. Our findings in HCM do not appear to involve these pathways and is consistent with different underlying pathophysiology for LVOT obstruction in HCM, as compared to the pathogenesis of aortic stenosis. The presence of an outflow tract gradient is thus not sufficient to account for the same proteomic changes seen in either condition. Nevertheless, 12 out of 79 proteins identified in our study were also found to be differentially expressed after surgery in the study by Aguado et al. [24]. This included 3 proteins increased after surgery (MMP12, RPS3, and CD5L) and 9 proteins decreased after surgery (HAMP, PLA2G1B, TNFSF12, MB, DCTN2, C3, FAM107B, ACP5, and BCL2L2).
Some study limitations warrant mention. Our study is limited by the relatively small sample size and the lack of an independent cohort to validate the results, which need confirmation; yet provides the first proteomic analysis of myectomy effects, and is strengthened by the fact that each case is its own control (post vs. pre). Given the sample size and the paired analysis, adjustment by Benjamini-Hochberg correction did not result in any significant proteins and false discovery, as the large number of proteins tested on a small number of samples might be a concern that requires confirmation in larger, independent cohorts.
Our study was the first to measure plasma proteomics in patients with obstructive HCM, before and after surgical myectomy, and to demonstrate potential pathogenic pathways affecting inflammation, fibrosis, and angiogenesis. Furthermore, our study identifies potential upstream regulatory therapeutic targets for LVOT obstruction in a human HCM population. Targeting of these putative upstream regulators might reduce inflammation, fibrosis, and angiogenesis, and thus might possibly be beneficial in the treatment of HCM, pending future validation studies.
A total of 12 patients with clinically documented HCM referred and scheduled for surgical myectomy were approached