Chronic activation of tubulin tyrosination in HCM mice and human iPSC-engineered heart tissues improves heart function.

Rationale: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that -α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Acute reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment. Objective: In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model ( Mybpc3 -knock-in; KI), in human HCM cardiomyocytes and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results: AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents and 3-week-old KI mice and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show that i) TTL for 6 weeks dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL for 12 weeks improved diastolic filling, cardiac output and stroke volume and reduced stiffness in KI mice; iii) TTL for 10 days normalized cell hypertrophy in HCM hiPSC-cardiomyocytes; iv) TTL induced a marked transcription and translation of several tubulins and modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-deficient EHTs exhibited reduced dTyr-tub levels, higher force and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion: This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

The W101C KCNJ5 Mutation Induces Slower Pacing by Constitutively Active GIRK Channels in hiPSC-Derived Cardiomyocytes.

Mutations in the KCNJ5 gene, encoding one of the major subunits of cardiac G-protein-gated inwardly rectifying K+ (GIRK) channels, have been recently linked to inherited forms of sinus node dysfunction. Here, the pathogenic mechanism of the W101C KCNJ5 mutation underlying sinus bradycardia in a patient-derived cellular disease model of sinus node dysfunction (SND) was investigated. A human-induced pluripotent stem cell (hiPSCs) line of a mutation carrier was generated, and CRISPR/Cas9-based gene targeting was used to correct the familial mutation as a control line. Both cell lines were further differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed GIRK channels which underly the acetylcholine-regulated K+ current (IK,ACh). hiPSC-CMs with the W101C KCNJ5 mutation (hiPSCW101C-CM) had a constitutively active IK,ACh under baseline conditions; the application of carbachol was able to increase IK,ACh, further indicating that not all available cardiac GIRK channels were open at baseline. Additionally, hiPSCW101C-CM had a more negative maximal diastolic potential (MDP) and a slower pacing frequency confirming the bradycardic phenotype. Of note, the blockade of the constitutively active GIRK channel with XAF-1407 rescued the phenotype. These results provide further mechanistic insights and may pave the way for the treatment of SND patients with GIRK channel dysfunction.

PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation.

BACKGROUND: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2-/-) in human induced pluripotent stem cell-derived atrial cardiomyocytes. METHODS: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. RESULTS: PITX2-/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2-/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2-/- versus isogenic control; P<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P<0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P=0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. CONCLUSIONS: Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

Piezo2 is not an indispensable mechanosensor in murine cardiomyocytes.

A short-term increase in ventricular filling leads to an immediate (Frank-Starling mechanism) and a slower (Anrep effect) rise in cardiac contractility, while long-term increased cardiac load (e.g., in arterial hypertension) decreases contractility. Whether these answers to mechanical tension are mediated by specific sensors in cardiomyocytes remains elusive. In this study, the piezo2 protein was evaluated as a potential mechanosensor. Piezo2 was found to be upregulated in various rat and mouse cardiac tissues upon mechanical or pharmacological stress. To investigate its function, C57BL/6J mice with homozygous cardiomyocyte-specific piezo2 knockout [Piezo2-KO] were created. To this end, α-MHC-Cre mice were crossed with homozygous "floxed" piezo2 mice. α-MHC-Cre mice crossed with wildtype mice served as controls [WT-Cre+]. In cardiomyocytes of Piezo2-KO mice, piezo2 mRNA was reduced by > 90% and piezo2 protein was not detectable. Piezo2-KO mice displayed no morphological abnormalities or altered cardiac function under nonstressed conditions. In a subsequent step, hearts of Piezo2-KO or WT-Cre+-mice were stressed by either three weeks of increased afterload (angiotensin II, 2.5 mg/kg/day) or one week of hypercontractility (isoprenaline, 30 mg/kg/day). As expected, angiotensin II treatment in WT-Cre+-mice resulted in higher heart and lung weight (per body weight, + 38%, + 42%), lower ejection fraction and cardiac output (- 30%, - 39%) and higher left ventricular anterior and posterior wall thickness (+ 34%, + 37%), while isoprenaline led to higher heart weight (per body weight, + 25%) and higher heart rate and cardiac output (+ 24%, + 54%). The Piezo2-KO mice reacted similarly with the exception that the angiotensin II-induced increases in wall thickness were blunted and the isoprenaline-induced increase in cardiac output was slightly less pronounced. As cardiac function was neither severely affected under basal nor under stressed conditions in Piezo2-KO mice, we conclude that piezo2 is not an indispensable mechanosensor in cardiomyocytes.

Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy.

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue.

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen.

Disease modeling of a mutation in α-actinin 2 guides clinical therapy in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α-actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient-derived human-induced pluripotent stem cells (hiPSCs) and show that hiPSC-derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L-type Ca2+ current. The L-type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM-affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease-causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof-of-principle for the use of hiPSC for personalized treatment of cardiomyopathies.

Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3.

The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel-based next-generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation-not only in adult-but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.

Author Correction: Differentiation of cardiomyocytes and generation of human engineered heart tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Biallelic mutation in MYH7 and MYBPC3 leads to severe cardiomyopathy with left ventricular noncompaction phenotype.

Dominant mutations in the MYH7 and MYBPC3 genes are common causes of inherited cardiomyopathies, which often demonstrate variable phenotypic expression and incomplete penetrance across family members. Biallelic inheritance is rare but allows gaining insights into the genetic mode of action of single variants. Here, we present three cases carrying a loss-of-function (LoF) variant in a compound heterozygous state with a missense variant in either MYH7 or MYBPC3 leading to severe cardiomyopathy with left ventricular noncompaction. Most likely, MYH7 haploinsufficiency due to one LoF allele results in a clinical phenotype only in compound heterozygous form with a missense variant. In contrast, haploinsufficiency in MYBPC3 results in a severe early-onset ventricular noncompaction phenotype requiring heart transplantation when combined with a de novo missense variant on the second allele. In addition, the missense variant may lead to an unstable protein, as overall only 20% of the MYBPC3 protein remain detectable in affected cardiac tissue compared to control tissue. In conclusion, in patients with early disease onset and atypical clinical course, biallelic inheritance or more complex variants including copy number variations and de novo mutations should be considered. In addition, the pathogenic consequence of variants may differ in heterozygous versus compound heterozygous state.

Gene therapy strategies in the treatment of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an inherited myocardial disease with an estimated prevalence of 1:200 caused by mutations in sarcomeric proteins. It is associated with hypertrophy of the left ventricle, increased interstitial fibrosis, and diastolic dysfunction for heterozygous mutation carriers. Carriers of double heterozygous, compound heterozygous, and homozygous mutations often display more severe forms of cardiomyopathies, ultimately leading to premature death. So far, there is no curative treatment against HCM, as current therapies are focused on symptoms relief by pharmacological intervention and not on the cause of HCM. In the last decade, several strategies have been developed to remove genetic defects, including genome editing, exon skipping, allele-specific silencing, spliceosome-mediated RNA trans-splicing, and gene replacement. Most of these technologies have already been tested for efficacy and efficiency in animal- or human-induced pluripotent stem cell models of HCM with promising results. We will summarize recent technological advances and their implication as gene therapy options in HCM with a special focus on treating MYBPC3 mutations and its potential for being a successful bench to bedside example.

Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes.

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

Differentiation of cardiomyocytes and generation of human engineered heart tissue.

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered heart tissue.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated.

Serum Matrix Metalloproteinases as Quantitative Biomarkers for Myocardial Fibrosis and Sudden Cardiac Death Risk Stratification in Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia (VT), and myocardial fibrosis reflects an important risk factor. Several matrix metalloproteinases (MMPs) and procollagen N-terminal propeptides (PNPs) are involved in collagen turnover and discussed as fibrosis biomarkers. We aimed to identify biomarkers that correlate with myocardial fibrosis in late-gadolinium-enhancement cardiac magnetic resonance imaging (LGE-CMR) and/or cardiac events (syncope, VT) in HCM patients. METHODS AND RESULTS: In 54 HCM patients (age 55.9 ± 14.3 y, 50% female) fibrosis was quantified by LGE-CMR. Serum concentrations of MMP-1, -2, -3, -9, and tissue inhibitor of MMP (TIMP) 1 were analyzed by means of enzyme-linked immunosorbent assay and PINP, PIIINP, and type I collagen C-terminal telopeptide (ICTP) concentrations by radioimmunoassay. MMP-9 was associated with fibrosis in LGE-CMR (mean increase 0.66 g/unit MMP9 [95% confidence interval [CI] 0.50-0.82]; P < .001) and with cardiac events in women (odds ratio [OR] 1.07 [1.01-1.12], P = .01) but not in men. Increased MMP-2 levels in women were associated with lower fibrosis (0.05 [-0.09 to -0.01]; P = .015). MMP-3 levels were positively associated with cardiac events (OR 1.13 [1.05-1.22]; P = .001) independently from fibrosis and sex. No association was detected for MMP-1, TIMP-1, PNPs, and ICTP. CONCLUSIONS: These data suggest that MMP-9 is a useful biomarker for fibrosis and cardiac events in female HCM patients, whereas MMP-3 is associated with a higher event rate independent from fibrosis and sex.

Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology.

More than 350 individual MYPBC3 mutations have been identified in patients with inherited hypertrophic cardiomyopathy (HCM), thus representing 40­50% of all HCM mutations, making it the most frequently mutated gene in HCM. HCM is considered a disease of the sarcomere and is characterized by left ventricular hypertrophy, myocyte disarray and diastolic dysfunction. MYBPC3 encodes for the thick filament associated protein cardiac myosin-binding protein C (cMyBP-C), a signaling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of contraction and relaxation. This review aims to provide a succinct overview of how mutations in MYBPC3 are considered to affect the physiological function of cMyBP-C, thus causing the deleterious consequences observed inHCM patients. Importantly, recent advances to causally treat HCM by repairing MYBPC3 mutations by gene therapy are discussed here, providing a promising alternative to heart transplantation for patients with a fatal form of neonatal cardiomyopathy due to bi-allelic truncating MYBPC3 mutations.