MYH11 rare variant augments aortic growth and induces cardiac hypertrophy and heart failure with pressure overload

Smooth muscle cell-specific myosin heavy chain, encoded by MYH11, is selectively expressed in smooth muscle cells (SMCs). Pathogenic variants in MYH11 predispose to a number of disorders, including heritable thoracic aortic disease associated with patent ductus arteriosus, visceral myopathy, and megacystis-microcolon-intestinal hypoperistalsis syndrome. Rare variants of uncertain significance occur throughout the gene, including MYH11 p.Glu1892Asp, and we sought to determine if this variant causes thoracic aortic disease in mice. Genomic editing was used to generate Myh11E1892D/E1892D mice. Wild-type (WT) and mutant mice underwent cardiovascular phenotyping and with transverse aortic constriction (TAC). Myh11E1892D/E1892D and WT mice displayed similar growth, blood pressure, root and ascending aortic diameters, and cardiac function up to 13 months of age, along with similar contraction and relaxation on myographic testing. TAC induced hypertension similarly in Myh11E1892D/E1892D and WT mice, but mutant mice showed augmented ascending aortic enlargement and increased elastic fragmentation on histology. Unexpectedly, male Myh11E1892D/E1892D mice two weeks post-TAC had decreased ejection fraction, stroke volume, fractional shortening, and cardiac output compared to similarly treated male WT mice. Importantly, left ventricular mass increased significantly due to primarily posterior wall thickening, and cardiac histology confirmed cardiomyocyte hypertrophy and increased collagen deposition in the myocardium and surrounding arteries. These results further highlight the clinical heterogeneity associated with MYH11 rare variants. Given that MYH11 is selectively expressed in SMCs, these results implicate a role of vascular SMCs in the heart contributing to cardiac hypertrophy and failure with pressure overload.


Introduction
MYH11 encodes the smooth muscle-specific isoform of myosin heavy chain (SMMHC), which associates with a regulatory light chain and a second light chain of unknown function and polymerizes to form the thick filament in the contractile unit of smooth muscle cells (SMCs) [1].
MYH11 is only expressed in SMCs, as illustrated by the fact that the Myh11 promoter is used as a Cre-driver to lineage-trace SMCs [2][3][4][5][6].Pathogenic variants in MYH11 confer a highly penetrant risk for several disorders, including heritable thoracic aortic disease associated with patent ductus arteriosus [7][8][9][10][11].Although rare missense variants are present throughout MYH11, the majority of pathogenic variants that cause heritable thoracic aortic disease are large, in-frame deletions in the coiled-coil domain, a region that is critical for polymerization of SMMHC into thick filaments.
Rare chromosomal duplications of 16p13.1 that include MYH11 and eight other genes also confer an increased risk for aortic dissection.However, there is no evidence that the corresponding deletion increases the risk for thoracic aortic disease (TAD) [12].Instead, recessive loss-offunction MYH11 pathogenic variants are responsible for fetal megacystis-microcolon.Finally, heterozygous variants that disrupt the termination codon at the C-terminus and add extra amino acids to the end of the protein predispose individuals to a smooth muscle dysmotility syndrome with esophageal, gastric, and intestinal complications [13][14][15].
MYH11 variants of uncertain significance (VUSs) are commonly reported with genetic testing, but the phenotypic variability and burden of MYH11 rare variants make it difficult to assign pathogenicity to identified variants.We previously determined that a VUS in MYH11, p.Arg247Cys (R247C), decreases myosin motor function in in vitro assays [16].Myh11 R247C/R247C mice show decreased aortic ring contraction, yet have normal growth, survival, and no evidence of TAD [16].However, when hypertension is induced using 3g/L L-N G -Nitro arginine methyl ester and a high-salt diet, one-fifth of Myh11 R247C/R247C mice die due to acute dissection of the proximal aorta (manuscript submitted).More recently, a heterozygous in-frame deletion in MYH11, p.Lys1256del, segregated with thoracic aortic dissection in two independent pedigrees [9].The homozygous p.Lys1256del mice also had no evidence of aortic disease up to 18 months of age, but aortic dissection rates are higher in both heterozygous and homozygous mutant mice with angiotensin II infusion compared to similarly treated wild-type (WT) mice [17].These data support that rare variants in MYH11 can contribute to increased risk for TAD.
MYH11 missense VUSs in the coiled-coil region have not been functionally assessed in mice, so we sought to determine if MYH11, p.Glu1892Asp (E1892D) increases the risk for TAD in a mouse model.Myh11 E1892D/E1892D mice did not develop TAD with age, but when male Myh11 E1892D/E1892D mice were subjected to transverse aortic constriction (TAC) for 2 weeks, augmented ascending aortic enlargement occurred when compared to WT mice.Unexpectedly, TAC also induced significant cardiomyocyte hypertrophy, increased cardiac fibrosis, and impaired left ventricular contractile function in the Myh11 E1892D/E1892D mice.These studies broaden our understanding of the phenotypes associated with MYH11 rare variants and identify a novel role for mutant arterial SMCs contributing to aberrant cardiac remodeling with pressure overload in mice.

Results
VUS in MYH11, p.Glu1892Asp, identified in a patient with TAD The proband is a 66-year-old male of European descent with an aortic root that progressively increased from 4.2 to 4.8 cm over 10 years, and he underwent a successful valvesparing aortic root and ascending aorta repair.The proband has pectus excavatum, pes planus, and myopia, but no other skeletal, cardiac, or ocular abnormalities.He also has hypercholesterolemia and hypertension that are controlled with medications.His father had a 4.2 cm aortic root and died of lung cancer at the age of 68 years, and his paternal grandfather died suddenly of an unknown cause at the age of 50 years.There was no other family history of TAD or sudden death.
Genome sequencing performed on DNA isolated from both peripheral blood leukocytes and tissues from the resected aorta showed no evidence of pathogenic variants in known aortopathy Pressure overload augments ascending aortic enlargement in We previously demonstrated that proximal aorta enlarges two weeks after TAC in WT C57BL/6J mice and is associated with aortic medial and adventitial thickening [18].
Myh11 E1892D/E1892D and WT mice of both sexes were subjected to TAC surgeries at 10-12 weeks of age.Mortality rates immediately following recovery from anesthesia were similar across the four groups: 22% (2/9) for male WT, 30% (3/10) for male mutants, 30% (3/10) for female WT, and 20% (2/10) for female mutants.These deaths were associated with acute congestive heart failure due to the constriction.Additionally, one female mutant mouse died of a ruptured left main coronary artery and cardiac tamponade one day after TAC, and one male mutant mouse died of congestive Heart tissue obtained from male WT and Myh11 E1892D/E1892D mice post-TAC underwent WGA staining [20,21].The cardiomyocyte cross-sectional area in the posterior wall of the left ventricle corroborated significant cardiomyocyte hypertrophy in the Myh11 E1892D/E1892D heart compared to WT hearts, while no difference was observed between the anterior walls (Fig 4D and

Discussion
A missense VUS in the coiled-coil domain of MYH11, p.Glu1892Asp, was identified in a proband with aortic root aneurysm.The functional impact of this variant was investigated by introducing it into the mouse genome.Similar to other mouse models of genetic variants predisposing to TAD [16,17], Myh11 E1892D/E1892D mice develop normally without thoracic aortic enlargement, but increasing the forces on the aorta via TAC augments ascending aortic enlargement in Myh11 E1892D/E1892D mice compared to similarly treated WT mice.MYH11, p.K1256del, is a pathogenic variant that causes an autosomal dominant inheritance of a predisposition for type A and B dissections [9].Although there is no evidence of TAD in mice heterozygous or homozygous for this variant, angiotensin II infusion induces both thoracic and abdominal aortic dissections in both heterozygous and homozygous mice [17].Thus, these data support that the MYH11, p.Glu1892Asp, variant increases the risk for TAD, but further data are needed to determine the penetrance and additional genetic or environmental factors that contribute to the penetrance of TAD associated with this variant.
An unexpected finding in this study is the sex-dependent aberrant cardiac remodeling observed in Myh11 SMCs, cardiomyocytes, and cardiac fibroblasts [23].
In this model, constriction of the transverse aorta leads to increased pressure load on the left ventricle, triggering a cascade of molecular events similar to those observed in clinical conditions such as poorly controlled hypertension or aortic stenosis.Studies utilizing the TAC mouse model to study the mechanisms underlying cardiac remodeling and pump failure need to consider the genetic background [24,25], degree and duration of constriction [26,27], and sex [28,29].Male C57BL/6J mice undergoing TAC using a 27-gauge needle develop cardiac hypertrophy and pump failure as early as 7 days after surgery, which are characterized by increased mass of left ventricle, thicknesses of septal and posterior wall, along with decreased ejection fraction and fractional shortening [26].In the current study, we replicate the decreased left ventricular contractile function in male WT mice 2 weeks after surgery using a 27-gauge needle [18], and identify further decline of heart contraction in male Myh11 E1892D/E1892D mice.It has been reported that TAC-induced cardiac hypertrophy and impaired contraction show sex differences 6 weeks after TAC in C57BL/6J WT mice [29].However, in this study, both ejection fraction and fractional shortening are significantly decreased in male versus female Myh11 E1892D/E1892D mice just 2 weeks after surgery, indicating a rapid decline of left ventricular contractile function in male When female mice lacking estrogen receptor beta gene (Esr2 -/-) are subjected to TAC for 2 weeks, a greater increase in heart weight relative to body weight is observed compared to WT littermate females [28].This finding suggests that estrogen receptor subtype beta plays a protective role in the development of pressure overload-induced cardiac hypertrophy and may be the mediator of observed sex differences.
We hypothesize that Myh11 E1892D/E1892D SMCs in coronary arteries may produce a signal that alters the cardiomyocyte.We previously identified increased IGF-1 expression in aortic tissue of a patient with MYH11 p.Leu1264Pro, another missense variant in the coiled-coil domain [8].
Transcriptomic analyses identified a 40-fold increase of IGF1 expression in the SMCs explanted from the patient's aorta compared to SMCs explanted from normal aortas.Thus, MYH11 rare variants may trigger excessive SMC IGF-1 production and be the source of SMC-tocardiomyocyte signaling to drive cardiac hypertrophy and failure in Myh11 E1892D/E1892D mice after TAC.One of the main pathways activated by IGF-1 is the PI3K/Akt/mTORC1 pathway that promotes protein synthesis and cell growth, contributing to cardiomyocyte hypertrophy in response to pressure overload.Inhibition of mTORC1 with rapamycin significantly attenuates cardiac hypertrophy and improves cardiac function with pressure overload [30,31].Activation of PI3K/Akt signaling could also lead to the phosphorylation and inactivation of the BCL2-associated agonist of cell death protein BAD and prevent oxidative stress-induced apoptotic death of cardiomyocytes, thereby enhancing cell survival [32].Additionally, IGF-1 signaling promotes angiogenesis, ensuring adequate oxygen and nutrient supply to the hypertrophied myocardium and supporting increased metabolic demands [33,34].However, if the underlying stress persists, chronic activation and maladaptive remodeling can eventually lead to heart failure.
Another interesting finding in this study is that pressure overload leads to increased cardiac fibrosis in the posterior wall of the Myh11 E1892D/E1892D hearts, characterized by increased periarterial and interstitial collagen deposition.Pressure overload-induced cardiac fibrosis is an intricate process influenced by various molecular mechanisms, with activated fibroblasts and myofibroblasts acting as the central effectors and serving as the main source of matrix proteins.
One key driver is the activation of the renin-angiotensin-aldosterone system due to decreased stroke volume and renal blood flow [35], facilitating fibroblast proliferation and collagen deposition in the myocardium [36,37].Increased biomechanical stress on the cardiac tissue leads to release and activation of transforming growth factor-beta signaling, stimulating fibroblast differentiation into myofibroblasts, which are responsible for excessive extracellular matrix production [38,39].The activation of profibrotic pathways, such as the renin-angiotensinaldosterone system and transforming growth factor-beta signaling, is evident in TAC-induced cardiac remodeling [37,40].Inflammatory responses mediated by cytokines and immune cells also contribute to the progression of fibrosis post-TAC [37], while oxidative stress and mitochondrial dysfunction have been implicated in TAC-induced cardiac fibrosis and dysfunction [41,42].
Additionally, fibroblasts can also become activated by mechanical stress through mechanosensitive receptors like integrins, ion channels, G-protein coupled receptors, and growth factor receptors and can activate downstream signaling pathways that promote matrix production [43].Further studies will define the predominant signaling pathway that mediates the rapid interstitial collagen deposition in male Myh11 E1892D/E1892D mice.
Genome-wide association studies (GWAS) have identified loci involving MYH11 associated with various traits of cardiac rhythm, including resting heart rate, heart rate response to exercise, atrial fibrillation, PR interval, and electrocardiography, suggesting a potential relationship between MYH11 variants and cardiac pacing and arrhythmias.Additionally, three genetic risk loci (rs216158, rs9972711, rs12691049) encompassing MYH11 have been linked to coronary artery disease [44].Notably, no loci linked to MYH11 have been associated with cardiac hypertrophy or heart failure in GWAS.
Collectively, these results demonstrate that a missense VUS in a gene almost exclusively expressed in SMCs, MYH11, does indeed increase thoracic aortic enlargement but also triggers aberrant pressure overload-induced remodeling of the heart that is characterized by increased cardiomyocyte hypertrophy, cardiac fibrosis, and heart failure in males.These findings provide further evidence of the diverse phenotypes associated with MYH11 rare variants and implicate vascular SMC-to-cardiomyocyte signaling in driving aberrant cardiac remodeling with pressure overload.Future studies will focus on the interactions among different cell types in the heart and identify specific cellular pathways downstream of the mutant contractile protein in SMCs that mediate SMC-cardiomyocyte communications and contribute to cardiomyopathy.

Animal study
All animal experimental procedures were designed in accordance with National Institutes of Health guidelines and approved by the Animal Welfare Committee and the Center for Laboratory Animal Medicine and Care at the University of Texas Health Science Center at Houston.Myh11 E1892D/+ breeders were transferred from the Jackson Laboratory and the colony was maintained on a C57BL/6J background.

Transverse aortic constriction surgery
At the age of 10-12 weeks, both male and female wild-type and Myh11 E1892D/E1892D mice were anesthetized by 0.3-0.5 L/min pure oxygen with 2% isoflurane and placed supine on a 38°C heating pad.Intubation was performed with a 22-gauge venous catheter connected to a rodent ventilator with a respiratory rate of 125-150 breaths/min and a tidal volume of 6-8 µL/g.Carprofen (dose of 5 mg/kg, subcutaneous injection) and lidocaine (dose < 2.25 mg/kg, subcutaneous injection) were administrated before an upper partial sternotomy incision (about 1cm) was made.
A 6-0 silk suture was coiled under the aortic arch between the innominate artery and the left common carotid artery and ligated with a 27-gauge needle placed by the aortic arch.The needle was then promptly removed to yield a constriction of 0.41mm in the outer diameter.The lungs were re-inflated before the skin was closed.Mice that died prior to the endpoint at fourteen days post-operation were subjected to necropsy to determine the cause of death.In male and female mice, comparable numbers succumbed following surgery (males: 2 out of 9 WT and 3 out of 10 mutants; females: 3 out of 10 WT and 2 out of 10 mutants).These expected post-operative mortality rates are primarily linked to acute congestive heart failure post-TAC [19], with the exception of one female that died due to coronary artery rupture (S1 Fig and S1 Table ).

Echocardiography
Echocardiography (Vevo 3100 imaging system, MX550D transducer, VisualSonics, Toronto, Canada) was performed two weeks post-surgery.Briefly, mice were weighed and anesthetized by 0.5-1.0L/min room air with 2% isoflurane via nose cone.Heart rate was closely monitored and body temperature was maintained around 38.5°C using the heating system.The aortic root and ascending aorta were imaged in B-mode.Left ventricular function derived from short axis parasternal planes was imaged in M-mode.Measurements of maximal internal diameter of the proximal aorta and left ventricular contractile function were obtained from three different cardiac cycles and averaged.Data were analyzed by an operator blinded to the treatment groups.

Invasive blood pressure measurement
Following echocardiography analyses, intraluminal blood pressure measurements were performed using a Millar pressure catheter (SPR-1000, 1.0F, Oakville, Ontario, Canada) inserted into the right common carotid artery.Mice were intubated and placed on a ventilator using the same conditions as in TAC surgery except replacing pure oxygen with room air.The 1.0F catheter was inserted into the ascending aorta to monitor the blood pressure.Stable pressure tracings were recorded for 5 minutes at a PCU-2000 pressure signal conditioner and PowerLab 4/35 station (ADInstruments Inc., Colorado Springs, CO, USA), and systolic and diastolic blood pressures were averaged from the midterm 4 minutes record.

Myographic assay of aortic rings
Ascending aortic tissues were harvested from both male and female mice at the age of 10 x 100, where DP is the maximal force pre-generated by PE, DD is the lowest force generated at a given dose of ACh or SNP and DB is the baseline force [45,46].

Histopathology
After intraperitoneal injection with Avertin (2.5%, 350 mg/kg), euthanized animals were perfusion fixed with 20 mL 1×PBS (pH=7.4)followed by 20 mL 10% neutral buffered formalin for 5 minutes through the left ventricle under physiological pressure.Ascending aortas and heart tissues were excised and further fixed in 10% neutral buffered formalin overnight at room temperature, then embedded in paraffin and sectioned at 5 μm.Aortic sections were stained with hematoxylin and eosin (H&E), Verhoeff Van Gieson (VVG, Polysciences, Inc., 25089-1), and Picro-Sirius Red (Abcam, ab150681) for morphometric analyses, medial elastic fibers and collagen content identification, respectively.Heart sections were stained with Picro-Sirius Red to determine collagen content.Images were obtained using a Leica DM2000 LED microscope, and analyzed with ImageJ software.Quantitative analyses were performed by 3 individuals blinded to the group information.

Wheat germ agglutinin staining
the project, secured funding, supervised the work, outlined and edited the manuscript.All authors read and approved the manuscript in its final form.
This research was funded by the Leducq Foundation, NIH HL146583, and private donations (D.M.M.).We would like to thank the family for participating in this study.We also grateful for the contributions of Nicholas Brown, Hongwei Jin, and the Center for Biometric Analysis at The Jackson Laboratory for expert assistance with this project.There is no difference of cardiomyocyte cross-sectional area between male wild-type and Myh11 E1892D/E1892D mice after TAC.ns, non-significant.• WT TAC, ■ Myh11 E1892D/E1892D TAC.S1 Table .Total numbers of mice died after transverse aortic constriction.

Supporting information
S2 Table .Segregation record of 102 progenies from heterozygous breeders.
heart failure on day eight (S1 Fig and S1 Table).Two weeks post-surgery, both male and female Myh11 E1892D/E1892D mice exhibited significant increases in the ascending aortic diameter compared to WT TAC mice, despite displaying comparable levels of systolic and diastolic blood pressure (Fig 3A-B and S2A-B Figs).Histology analysis revealed significant increases in medial thickening and the number of elastic breaks in the mutant aortas compared to WT aortas, with no difference in adventitial area or collagen accumulation (Fig 3A and 3C).Pressure overload induces left ventricular posterior wall hypertrophy and heart failure in male Myh11 E1892D/E1892D mice TAC increases cardiac afterload and is routinely used to study cardiac hypertrophy and heart failure[19].Unexpectedly, male Myh11 E1892D/E1892D mice undergoing TAC had significantly impaired left ventricular contractile function by echocardiographic studies two weeks after TAC, as illustrated by the decreased ejection fraction, stroke volume, fractional shortening, and cardiac output in these mutant mice compared to similarly treated male WT mice (Fig4A-B); these changes were not present in the female mutant mice compared to female WT mice (S2C Fig).Subsequent evaluation revealed that TAC induced a significant increase in left ventricular mass in male Myh11 E1892D/E1892D mice compared to male WT TAC mice, primarily characterized by posterior wall thickening (Fig4C).Additionally, both end-systolic and end-diastolic diameters and volumes of the left ventricle were significantly enlarged in male Myh11 E1892D/E1892D mice (Fig4C).In contrast, alterations of these cardiac remodeling were not observed in female Myh11 E1892D/E1892D mice after TAC, except those limited exclusively to the left ventricular enddiastolic diameter when compared to WT female mice (S2D Fig).

S3
Fig).Quantification for collagen deposition showed increased peri-arterial and left ventricular posterior wall fibrosis in the Myh11 E1892D/E1892D hearts compared to WT group (Fig 4E).
months and delivered in ice-cold Hanks' Balanced Salt Solution through overnight shipping, and then cut into 2-mm ring segments and placed in the 620M Multi Chamber Myograph System (Danish Myo Technology, Hinnerup, Denmark) filled with 8 mL of oxygenated (95% O2, 5% CO2) physiological saline solution (118.31 mM NaCl, 4.69 mM KCl, 1.2 mM MgSO4, 1.18 mM KH2PO4, 24.04 mM NaHCO3, 0.02 mM EDTA, 2.5 mM CaCl2, and 5.5 mM glucose) and allowed to equilibrate at 37 °C for at least 30 min.Aortic rings were stretched in 2-4 mN increments from 0 mN until the calculated transmural pressure reached 13.3 kPa (100 mmHg).Optimal resting tension was applied to the rings based on the passive vascular length-tension relationship.Cumulative concentration-response curves to phenylephrine (PE, 10 −9 to 10 −5 M) and potassium chloride (KCl, 5-100 mM) were generated to assess contractile function.Vascular relaxation was assessed with acetylcholine (Ach, 10 −9 to 10 −5 M) and sodium nitroprusside (SNP, 10 −9 to 10 −5 M) administration.Concentration-response curves to Ach and sodium nitroprusside were generated after rings were pre-constricted to 70% of maximum with PE.Doses were added after the response curve reached a plateau from the previous dose.Percent vasocontractile responses (%) were calculated for PE and KCl as [(DP − DB)/DB] × 100, where 'DP' is the maximal force generated by a given specific dose and 'DB' is the baseline force.Percent relaxation responses were calculated as [(DP -DD)/ (DP -DB)]

Fig 2 .
Fig 2. Assessment of myograph and smooth muscle cell contractile protein expression in

Fig 3 .
Fig 3. Assessment of ascending aortic remodeling 2 weeks after transverse aortic constriction

Fig 4 .
Fig 4. Assessment of left ventricular remodeling 2 weeks after transverse aortic constriction

S4Fig.
Comparison of left ventricular contractile function between male and female mutant mice 2 weeks after transverse aortic constriction (TAC).Male Myh11 E1892D/E1892D mice exhibit significantly lower ejection fraction and fractional shortening compared to female mutant mice following TAC.ns, non-significant; ** P<0.01.
genes, but revealed a VUS in MYH11, p.Glu1892Asp (c.5676G>C; CADD score 23.70 and REVEL score 0.562).The variant is located at the C-terminus in the α-helical coiled-coil domain and occurs in the gnomAD database at a frequency of ~0.6% in European populations, ~0.2% in South Asian and African/African-American populations, and is not found in East Asian populations.The relatively high frequency of the variant led to the categorization as benign or likely benign in Clinvar (VCV000138358.34).
[2][3][4][5][6]n is the most specific marker of SMCs identified to date and it is not expressed in other cell types, including myofibroblasts[2][3][4][5][6].Thus, our data indicate that a rare variant in a gene expressed exclusively in SMCs can trigger increased cardiomyocyte hypertrophy and decreased left ventricular contractile function, implicating a novel role for arterial SMCs in driving pathologic cardiac remodeling with pressure overload, in this case in a sex-specific manner.Pathogenic variants in FBN1, which encodes fibrillin-1, a protein that is a major component of extracellular matrix microfibrils, are the cause of Marfan syndrome, a genetic disorder characterized by TAD, skeletal, and ocular abnormalities.Although pathogenic variants in FBN1 are associated with an increased risk for dilated cardiomyopathy in both patients and mice, FBN1 is expressed in many tissues, including [22]2D/E1892D mice with pressure overload, as evidenced by the increased cardiomyocyte hypertrophy, posterior wall thickening and left ventricle failure following TAC.TAC is a well-established model to mimic hypertensive heart failure in humans, particularly replicating cardiac hypertrophy and subsequent heart failure[22].