A Perspective on Personalized Therapies in Hypertrophic Cardiomyopathy

Abstract

A dominant mechanism of sudden cardiac death in the young is the progression of maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy (HCM). Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. We discuss approaches for personalized treatments that are indicated by recent advanced understanding of the progression

Introduction

A dominant mechanism of sudden cardiac death in the young is the progressive maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy (HCM)^1–3. Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. The result is a disturbance in the control of cardiac function at the level of the sarcomere, which we have recently reviewed⁴,and which causes an increase in myofilament tension when activated by Ca²⁺. Depending on the mutation, the heart responds with progressive delayed relaxation, hyper-contractility, maladaptive mechano-transduction, and hypertrophic remodeling^5–7. We have also summarized a potential role of vascular abnormalities in HCM progression⁸. Figure 1 summarizes this progression in a scheme emphasizing the diversity of carriers of the HCM mutations as well as the complex pathways progression can take. The complexity of clinical management of HCM has been extensively considered ^9,10. As an example of this complexity, in Table 1 we have compared published retrospective data describing the differences in the general clinical course of symptomatic patients with HCM linked to mutations in either thin or thick filament proteins.^{11, 12} With thin filament mutations, there was a greater occurrence of progressive NYHA Class IV heart failure, with LVEF <50%, restrictive diastole, and severe atrial dilation and arrythmias. There was a greater occurrence of septal hypertrophy with thick filament mutations, whereas thin filament mutations exhibited apical concentric hypertrophy. Current therapies listed follow guidelines for therapeutic approaches related to clinical phenotype of atrial arrhythmias and symptomatic heart failure. Table 1 also lists therapy with Mavacamten, a myosin-directed molecule, which has demonstrated efficacy as a sarcomere and tension inhibitor in a phase 3 clinical trial (EXPLORER-HCM) treating patients with obstructive HCM^{13, 14}. Treatment with Mavacampten, improves exercise capacity, outflow track obstruction, and NYHA heart failure class, but does not appear to prevent progression¹⁴. Treatment of patients with Ranolaxine or GS-967, both of which are late Na current inhibitors have also induced a reduction of the high tensions prevalent in the septum in patients with obstructive HCM¹⁵. Ranolazine may also act to depress enhanced myofilament response to Ca²⁺¹⁶.

Figure 1. Progression of disease in carriers of HCM mutations.

The scheme emphases the diversity of carriers as well as the complexity and diversity of pathological progression leading to outflow obstructions, heart failure, and in some cases to sudden cardiac death (SCD). These pathological signatures are triggered by the biophysical defect imposed by the mutation, but what happens next may involve a clinical course along the divergent pathways contributing to diseases via the different mechanisms outlined. Although microcirculatory disorders occur in HCM, understanding of the involvement of the blood and lymphatic vasculature and their endothelial and mural cell components are understudied, and thus poorly understood. See text for discussion of current approaches to risk stratification based on translational control mechanisms

Table 1. Comparison of triggering mutations in thin and thick filament proteins in the clinical course and therapies in HCM.

See text for sources of data, references and further discussion.

Trigger/Clinical Course	Thin Filament Mutation	Thick Filament Mutation
Likely Biophysical Trigger	Increased Myofilament Ca-Response; Slowing of cross-relaxation kinetics Tension Cost (↑↑↑)	Increase in cross-bridge relaxation kinetics: Enhanced DRX Tension Cost (↑↑↑)
Adverse Function	Diastolic Dysfunction (↑↑↑) Triphasic LV Filling (↑↑↑) Outflow Track Obstruction (↑)	Diastolic Dysfunction (↑↑) Triphasic LV Filling (↑) Outflow Track Obstruction (↑↑↑) Hyper-contractility Increased Systolic Elastance
Early Adverse Remodeling	Apical Concentric Hypertrophy  LV Mass (↑)  Fibrosis (↑↑↑)	Septal Hypertrophy (↑↑↑) LV Mass (↑↑) Fibrosis (↑↑)
Late Adverse Remodeling	Restrictive Evolution (↑↑↑) Symptomatic Heart Failure (↑↑↑)	Hypokinetic Evolution (↑↑) Symptomatic Heart Failure (↑)
Altered E-C Coupling	Atrial /Ventricular Fibrillation Altered Ca-fluxes Sudden Cardiac Death (↑↑↑)	Atrial /Ventricular Fibrillation Altered Ca-fluxes Sudden Cardiac Death
Altered Energetics	Tension Cost (↑↑) Oxidative Stress (↑)	Tension Cost (↑) Reductive Stress (↑) Mitochondrial Abnormalities Mismatch of ATP demand
Therapies
Invasive	ICD Atrial Catheter Ablation	ICD Atrial Catheter Ablation Septal Myectomy
Pharmacological	β-blockers Verapamil (++) Diltiazem Amiodarone Diuretics (+++) ACE Inhibitors (+++) ARBS (+++) Disopyramide	β-blockers Verapamil Diltiazem (+++) Amiodarone Diuretics ACE Inhibitors (+) ARBS (+) Disopyramide (+++) Ranolazine (Late Na current Inhibitor
Phase 3 clinical trial		Sarcomere Inhibitor (Mavacampten)

Data in Table 1 highlight unmet needs for patients progressing to serious cardiac disorders. One is the need for early diagnosis, stratification, and management. Early interventions in mouse models have demonstrated the prevention of progression^{5, 7}. Some success at using various imaging techniques and strain measurements¹⁷ and exercise testing¹⁸ for stratification show promise. Compared to controls, patients harboring cTnT mutations and tested with cardiac magnetic resonance and [11C]-acetate positron emission tomography imaging demonstrated inefficiencies in myocardial oxygen consumption that occurred before mechanical dysfunction¹⁹. In the case of prevention of malignant arrhythmias, Maron B, Maron M. and colleagues^20–22 have made a strong case for the implementation of a combination of relevant demographic and clinical risk variables together with physician experience, knowledge, and intuition in the decision of when and whether to implant an ICD. A second need is to broaden the diagnosis in relation to precision medicine in understanding the endophenotype¹. A third is the need to determine early changes in the vascular and lymphatic compartments related to ischemia, fibrosis, and inflammation. Whereas alterations in the micro-circulation are documented in HCM, little is known of the evolution and impact of these changes²³.

State of the art approaches aim to develop prognostic early biomarkers and therapeutic targets tailored to individuals.

An emerging approach to individualized therapies in precision medicine includes profiling mRNA and micro-RNA in heart tissue of animal models and of patients with different causal mutations^{11, 24, 25}. In a recent example of this approach Vakrou et al.¹¹ compared 5 week old (adolescent) mouse models with a mutation in the myosin heavy chain (R403Q-αMHC) and a mutation in cardiac troponin T (R92W-cTnT). Biophysical triggers are different in these two models, and although some similarities are indicated as listed in Table 1, the cTnT mutation induces diastolic abnormalities, whereas the MHC mutation does not. The experiments tested the hypothesis that progression to symptomatic HCM and sudden death involves different signaling pathways. Measurements included determination of differences in the transcriptome, miRNAome, as well as the redox environment with emphasis on mitochondrial generated reactive oxygen species. The data indicated significant elevations in oxidative stress in the R92W-cTnT hearts compared to hearts of littermates and R403Q-αMHC-mice, which demonstrated reductive stress with elevated levels of reduced glutathione. Reductive stress also drives cardiac remodeling.²⁶ We have previously provided evidence that oxidative-stress related S-glutathionylation of myosin binding protein C (MyBPC3), slows cross-bridge kinetics and is likely to be responsible in part for the differences in diastolic function in the R92W-cTnT hearts^{16, 27–29}. Vakrou et al.¹¹ also reported an upregulation of the gene encoding the serpine1. Below we discuss the possible role of serpins, which were named for their activity as serum protease inhibitors. Serpins are large family of proteins that functions in protein quality control, processing of pro-matrix metalloproteinase 9, control of cell death, as well as inflammatory and infectious diseases.^30–32,33 Bioinformatic analysis of the omic data revealed that the cTnT mutation was more closely associated with oxidative stress related fibrotic remodeling via the TGF-β pathway. These data indicated the use of anti-fibrotic angiotensin receptor blockers (ARBs) such as losartan as a basis for personalized early treatment of individuals harboring the R92W-cTnT mutation. It is also relevant that agents reducing oxidant stress and elevating reductive stress are not appropriate for use in the MHC mutant mice. In view of reduced levels of peroxisome proliferator-activated receptor-γ (PPARγ) and increased inflammatory signaling in the MHC mutants, pathway analysis indicated the use of prosiglitazone, which activates PPARγ and reduces inflammation.³⁴ Thus, determinations of mRNA signatures contributed to understanding of the predominant clinical course as well potential interdictions to HCM progression.

More recently, Bos et al.²⁵ compared mRNA profiles from healthy donor human heart samples and from myectomy samples from patients with HCM. In this case comparisons were made of major genotype groups expressing mutant cMYBP-C, likely to be mostly truncation mutants, and mutations in the myosin heavy chain (MHC-HCM). Tissue samples harvested from septal myectomies meant that obstructive HCM was common to both disease-causing genes. Thus, the transcriptomes of these two HCM cohorts were not analyzed separately. In HCM the most up-regulated gene compared to controls encodes angiotensin converting enzyme 2 (ACE2). As discussed below section, the most down-regulated gene encodes Serpin A3, Inasmuch as binding of virus to ACE2 is a triggering event in SARS-CoV-2 infection^{35, 36}, Bos et al.²⁵ emphasized the implications of this increase in ACE2 in myocardial injury in infected patients. A limitation of the study is that myectomy is a procedure of last resort to relieve obstruction to ventricular flows. Thus, it was not possible to make conclusions regarding whether increased levels of ACE2 were present mainly at this disease endpoint and whether they occurred in the cardiac myocytes. This last concern was addressed in recent study by Tucker³⁷ et al, using single nucleus RNA-Seq (snRNA-Seq) analysis on biopsies from non-failing, HCM and DCM patients. While ACE2 expression predominates in cells associated with the microvasculature (pericytes), vascular smooth muscle cells and fibroblasts in non-failing hearts, its expression profile is significantly decreased in these cellular populations of HCM and DCM patients, but more importantly, was found to significantly increase in the cardiac myocytes of HCM and DCM patients³⁷. Thus, the implications of ACE2 expression changes in inherited cardiomyopathies in the context of the present COVID-19 pandemic bear comment, especially considering the therapeutic challenges associated with treating HCM, a disease of the cardiac myocyte with different genetic origins.

SARS-CoV-2 and Hypertrophic Cardiomyopathy

There is a growing recognition among the medical community at the forefront of tackling this pandemic, that the clinical management of patients with inherited cardiomyopathies and channelopathies needs to be prioritized differently with respect to COVID-19 than other forms of heart failure. A recent perspective by Limongelli and Crotti, lists COVID-19 medications in present use and their potential side effects in patients with cardiomyopathies³⁸. This is reinforced by case reports of patients infected with SARS-CoV-2 showing post-mortem microscopic findings of putative viral particles inside various cellular and extracellular compartments of the heart, including the cardiac myocytes³⁹. There are studies reporting high levels of virus not in myocytes, but cells of the endothelial compartment and pericytes^{40, 41}, However, these studies were not carried out in hearts of HCM COVID-19 patients. As noted above, there is increased ACE2 expression in the cardiac myocytes of HCM patients, which could induce different mechanisms of cardiac injury for HCM patients with COVID-19. Some insight into these potential mechanisms have been provided by infecting SARS-CoV-2 into cardiomyocytes derived from human inducible pluripotent stem cells (hiPSC-CMs). SARS-CoV-2 infection of hiPSC-CMs induced a cessation of beating, cytotoxicity and cell death⁴². Internalization of the virus could be diminished by pre-incubation with an ACE2 antibody. Moreover, viral mediated transcriptomic changes showed downregulation of genes associated with metabolism, mitochondrial and contractile function, whereas genes associated with cytokine production were upregulated. Of note, ACE2 was downregulated in the infected myocytes which would be important to compare to iPSC derived cardiomyocytes from HCM patients which appear different ACE2 expression profiles as noted. Thus, while these data obtained in cell-based assays provide insight into the potential pathophysiologic consequences associated with SARS-CoV-2 infection in cardiac myocytes, they could be leveraged to study HCM patient specific hiPSC derived cardiomyocytes.

Serpins and HCM

An issue not explored to our knowledge is the potential significance of alterations in translation of genes encoding serpins in HCM. As summarized above, Volkou et al.¹¹ reported that both the cTnT mutation in mice showed increases in Serpine1 compared to control litter mates. In contrast, Bos et al.²⁵ reported that in human myectomy samples from heart with outflow obstructive HCM, Serpin A3 was the most downregulated gene. This difference in transcription of serpin genes may be of significance in understanding the difference in the clinical course of HCM mutations. The HCM cTnT mutations have a predominant fibrotic signature, and Serpine1 encodes plasminogen activator inhibitor (PAI), which is a well-known regulator of fibrosis in multiple tissues⁴³. Although PAI-1 has been shown to repress cardiac fibrosis, studies reported by Flevaris et al.⁴⁴ demonstrated PAI-1 can also lead to fibrosis in the myocardium by its specifically promoting TGF-β production. In the case of HCM linked to the MHC mutations, the clinical course indicates a predominant inflammatory signature in the mouse studies of Volkou et al. The MHC mutations progressing to outflow track obstruction are associated with down-regulation of cardiac Serpin A3²⁵. SerpinA3 is a serine protease inhibitor with multiple functions including maturation of pro-matrix metalloproteinase 9, regulation of apoptosis, infectious diseases, and inflammation.^30–33 SerpinA3, known as anti-chymotrypsin or ACT, is an important inhibitor of neutrophil proteases, with cathepsin G as a major target⁴⁵. In the heart cathepsin G, one of a class of inflammatory serum proteases (ISPs), activates inflammasome pathways and promotes pathological remodeling in the heart^{46, 47}. Cathepsin G activity is important in cardiac repair after injury by adaptive proteolysis of dead cells and extracellular matrix. However, elevations in cathepsin G have been shown to activate the inflammasome and promote maladaptive remodeling in the uninjured heart. Hooshdara et al.⁴⁶ reported inhibition of cathepsin G reduces myocyte death and improves remodeling after ischemia reperfusion injury. It may also be of significance that cathepsin G is immobilized on arterial endothelium with functions important in arterial myeloid cell recruitment with specific effects on arterial leukocyte adhesion. Thus, the imbalance between SerpinA3 levels and cathepsin G represent a maladaptive effect in HCM, and this imbalance is likely to exacerbate the effects of elevations of ACE 2 in HCM with the inflammatory processes in SARS-CoV-2 infections.

Transcriptional profiling in HCM

In summary, these recent publications provide evidence of the value of global mRNA profiling in HCM, a common cause of sudden death. There is a growing need to target the determination of the cell-specific transcriptome in the cardiac myocyte and the arterial endothelium in HCM. As such, the continual development and application of new and exciting approaches such as single-cell and single nucleus RNA-Seq, mRNA ORF ribosomal foot printing and ribosome affinity purification (TRAP) to isolate the cell-type actively translating mRNAs, offer greater in-depth cell-type specific and cellular resolution of transcriptomic profiles. Technical issues related to cardiac myocyte dissociation has largely stifled scRNA-Seq progress in the adult heart but have not precluded analysis of embryonic and neonatal hearts. Moreover, recent studies have selectively sacrificed the cardiac myocytes and have focused on characterization of non-myocyte cell populations in the adult heart in consideration of their contribution to heart disease. Of note, Li et al, used endothelial cell specific lineage tracing models to demonstrate distinct transcriptional profiles and pathways through which endothelial cells enhance neovasculogenesis in the heart following ischemic injury⁴⁸. Despite exciting progress in scRNA-Seq approaches in the heart, the application to HCM remains elusive even in rodent HCM models. Ultimately, single nuclear RNA-Seq (snRNA-Seq) may provide the translational bridge into examining individualized cell type specific transcriptomic profiles since it circumvents cell isolation/labeling issues and holds the potential to isolate nuclei from frozen human biopsies^{49, 50}. However, mRNA ORF ribosomal foot printing and ribosome affinity purification (TRAP) hold the possibility of examining post-transcriptional and co-translational regulation and to move beyond the use of transcriptomic profiling to help predict pathways and signaling networks, to identifying the actively translating proteome within pathways. The power of mRNA ORF ribosomal foot printing has already been demonstrated by its ability to infer evidence of protein truncations and microproteins in human dilated cardiomyopathic hearts but does not provide direct analysis of the cell-type translating proteome⁵¹. However, extension of the ribosome affinity purification (TRAP) approach whereby cell-type specific RiboTag reporter mice are crossed with HCM models, holds the possibility to combine simultaneous ribosome affinity purification of the actively translating mRNAs and proteins within the different cellular compartments^{52, 53}.

In case of HCM patients infected with SARS-CoV-2, there is a threat to these translational mechanisms. The reliance of viruses on the host’s transcriptional and translational machinery for propagation and their capacity to manipulate mechanisms such as impairing cap-dependent ribosomal recruitment of host mRNA and targeting translation factor function may alter the balance of translation in favor of viral protein synthesis.⁵⁴ In the context of HCM in which transcriptional and translational events are already enhanced to drive structural and functional remodeling, is it worth considering the possibility of detrimental viral interference of protein synthesis as a secondary insult to the host’s immune response, as well as the loss of general maintenance of cellular integrity. Thus, the application of these emerging “omics” approaches for discovery purposes and the potential to harmonize data from the actively translating mRNA and proteins pools, not only offers a more refined understanding of individualized cell-type and/or mutation specific therapies for HCM disease progression, but also the development of more effective means for diagnosing and treating those cells and organs infected by SARS-CoV-2.