Introduction Heart failure (HF) remains a major clinical challenge, with limited therapeutic strategies directly targeting cardiac myocyte dysfunction. Drugs which activate cGMP-dependent protein kinase 1(PKG1), such as sacubitril/valsartan, nitrates, and vericiguat, improve LV function and outcomes in HF but are limited by PKG1-induced vasodilation and hypotension. Mixed-lineage kinase 3 (MLK3) is a novel substrate of PKG1α which mediates PKG1 preservation of LV function in heart failure models, yet does not mediate the acute hypotensive effects of PKG1 activation. While MLK3 kinase activity enhances contractility, its therapeutic potential in HF remains unexplored. This study investigated whether AAV9-mediated MLK3 overexpression after transaortic constriction (TAC) restores LV function and opposes pathological LV remodeling in a preclinical heart failure model while bypassing PKG1α-induced hypotension. Hypothesis AAV9-mediated MLK3 overexpression in a pressure-overload HF model preserves LV systolic and diastolic function and opposes myocardial fibrosis. Methods Male 12-week-old mice underwent 26G TAC or sham surgery. Five days post-TAC, AAV9-MLK3 or AAV9-TdTomato (negative control) was delivered via retro-orbital injection under the cardiac myocyte-specific cTNT promoter. Cardiac function was evaluated 28 days later using echocardiography, invasive hemodynamics, and histological analysis. Results At day 5 post-TAC, compared with sham, mice subjected to TAC developed reduced LV ejection fraction, prior to AAV9 randomization. At 28 days post-AAV9, the AAV9-MLK3-treated TAC mice exhibited significantly improved LV ejection fraction and fractional shortening compared to TAC controls. MLK3 overexpression preserved LV systolic pressure and enhanced diastolic indices, including mitral E/A ratio, tau, and LV relaxation rate. No significant changes were observed between AAV9 groups in LV wall thickness, whole heart mass, or LV mass normalized to tibia length. Histological analysis revealed persistent fibrosis in both MLK3-treated and control TAC mice, indicating that MLK3 functional benefits occur independently of fibrosis modulation. MLK3 expression was increased approximately 3-fold in AAV9-MLK3 LV tissue compared with control AAV9. Conclusion AAV9-mediated MLK3 overexpression significantly improves LV function in pressure-overloaded hearts but does not reduce fibrosis. This supports that MLK3 enhances cardiac performance through fibrosis-independent mechanisms, such as direct effects on cardiomyocyte contractility or intracellular signaling. While MLK3 activation presents a promising therapeutic avenue for HF, additional strategies may be needed to mitigate fibrosis-related disease progression.

A common form of heart failure improves when cyclic guanosine monophosphate-dependent protein kinase is activated with a novel agent that promotes cysteine-42 oxidation.

Background:Heart failure with preserved ejection fraction (HFpEF) is a major clinical challenge characterized by diastolic dysfunction. Left ventricular stiffening and inflammation are hallmarks of HFpEF, yet the contribution of extracellular matrix (ECM) stiffness and the immune-stromal mechanisms driving ECM stiffening in cardiometabolic HFpEF remain poorly understood. Methods:We used the murine "2-hit model" of cardiometabolic HFpEF, in which the combination of high fat diet and hypertension induced by L-NAME causes diastolic dysfunction. We evaluated diastolic function by echocardiography and ECM mechanics by uniaxial tensile testing of decellularized cardiac tissue. Functional in vivo studies included genetic depletion of T cells, interferon-γ (IFNγ) knockout mice, and pharmacological lysyl oxidase inhibition. We combined co-cultures of CD4+ T cells and cardiac fibroblasts (CFB) with mechanical testing of cardiac ECM and molecular biology to elucidate cellular and molecular mechanisms. Results:Left ventricular ECM stiffness strongly correlated with impaired diastolic function in experimental cardiometabolic HFpEF. Cardiac CD4+ T cell infiltration was required for ECM stiffening and upregulation of lysyl oxidase enzymes in CFB. CD4+ T cell-derived IFNγ was both necessary and sufficient to induce LOXL3 in CFB, which increased ECM stiffness in vitro. Mechanistically, IFNγ signaling activated hypoxia-inducible factor-1α (HIF1α) in CFB, driving LOXL3 expression and subsequent collagen crosslinking. Genetic or pharmacologic disruption of this IFNγ-HIF1α-LOXL3 axis in vivo attenuated adverse ECM remodeling and improved diastolic function. Conclusions:CD4+ T cells promote pathological ECM stiffening in cardiometabolic HFpEF through IFNγ-mediated, LOXL3-dependent ECM crosslinking by CFB. Targeting this immune-stromal pathway may offer a novel therapeutic strategy for HFpEF.

Heart failure (HF) remains a leading cause of morbidity and mortality worldwide, driven by diverse pathophysiological mechanisms. Among its major risk factors, obesity has emerged as a lobal public health concern affecting individuals across all age groups. The rising prevalence of obesity significantly increases the risk of cardiovascular complications, including the development and progression of HF. MicroRNAs (miRNAs), small non-coding RNA molecules, have garnered attention for their regulatory roles in cardiovascular disease, particularly through post-transcriptional modulation of gene expression. This review highlights the involvement of miRNAs in key pathological processes observed in the obese heart, including cardiac remodeling, apoptosis, angiogenesis, inflammation, mitochondrial dysfunction, and myocardial lipotoxicity. Understanding how specific miRNAs and their targets contribute to HF in the context of obesity may inform the development of novel RNA-based therapeutic strategies for cardiometabolic disease.

BACKGROUND:Type 2 diabetes mellitus (DMII) and metabolic syndrome (MBS) increase ventricular arrhythmia and sudden cardiac death risk. OBJECTIVE:This study aimed to identify mechanisms through which DMII and MBS promote ventricular tachycardia (VT). METHODS:We performed programmed ventricular stimulation on leptin receptor mutant (Db/Db) mice with DMII; high-fat, high-sucrose (HFHS) diet-fed mice with MBS; and cyclic guanosine monophosphate (cGMP)-dependent protein kinase 1 alpha (PKG1α) leucine zipper mutant (LZM) mice, which have neither DMII nor MBS but have disrupted PKG1α signaling. RESULTS:Programmed ventricular stimulation induced VT in Db/Db and HFHS diet-fed mice. Both models demonstrated autonomic dysfunction and decreased cGMP owing to decreased cardiac parasympathetic responsiveness. Conversely, cGMP augmentation with soluble guanylate cyclase stimulation (riociguat) or phosphodiesterase 5 inhibition (sildenafil) reduced VT inducibility. PKG1α LZM mice had normal autonomic responsiveness, but excess VT. Left ventricular tissue from HFHS diet-fed and LZM mice demonstrated hyperactivated glycogen synthase kinase-3 beta (GSK3β). GSK3β inhibition with TWS119 abolished inducible VT in all 3 models. Sarcoplasmic reticulum Ca2+ reuptake was delayed in cardiomyocytes (CMs) from all 3 models, reflected by increased time constant of cytoplasmic Ca2+ decline, tau. TWS119 reversed this effect. Phospholamban, which when unphosphorylated inhibits sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a-mediated Ca2+ reuptake from the cytosol, was hypophosphorylated in HFHS diet-fed and LZM mice, which was reversed by TWS119. CMs from HFHS diet-fed mice displayed increased frequency of early afterdepolarizations, further supporting Ca2+ dyshomeostasis. cGMP augmentation with riociguat/sildenafil prevented afterdepolarizations in HFHS CMs. CONCLUSION:In DMII and MBS, reduced PKG1α signaling drives GSK3β hyperstimulation, calcium dyshomeostasis, and VT. Pharmacologic modulation of these pathways opposes VT pathogenesis.

Introduction: Combined obesity and hypertension alter the endoplasmic reticulum (ER) stress response by downregulating X-box binding protein 1 ( Xbp1 ) in T cells, which heightens T cell inflammatory potential and persistence in Heart Failure with Preserved Ejection Fraction (HFpEF), a prevalent syndrome with no cure. The ER protein Stimulator of Interferon Genes (STING), in concert with Xbp1 , coordinates T cell inflammation and death in other contexts, yet its role in the T cell inflammatory response in cardiometabolic HFpEF is unknown. Hypothesis: STING activation and dysregulated ER stress responses drive T cell inflammation in HFpEF by balancing T cell activation and persistence in response to combined comorbidities. Methods: C67BL/6J (wild type, WT) mice were fed high-fat diet/L-NAME (H/L) or standard chow (STD) for 5 weeks, and STING activation was measured in splenic CD4 + T cells by Western blotting. Splenic CD4 + T cells from WT or STING-deficient mice (STING -/- ) were activated with αCD3/αCD28 for 3 days in vitro , then treated with complete media (CM) or CM with 0.2 mM palmitate (abundant in dietary fat)/0.2 mM L-NAME (PALNA) for up to 48 hours. T cell gene expression, activation, and death were assessed using qPCR, flow cytometry, Western blotting, and Lionheart live-cell microscopy. Results: Mice fed H/L had significantly increased splenic T cell expression of phospho-STING, alongside decreased T cell Xbp1 expression and increased cardiac inflammation, compared to STD-fed mice. Ex vivo incubation of CD4 + T cells with PALNA significantly decreased Xbp1 gene expression and increased apoptosis, indicated by cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expression, compared to cells in CM. However, the remaining live PALNA-treated cells showed higher expression of the activation protein CD69, and this was sustained over time, compared to cells in CM. Strikingly, live cell imaging revealed that STING -/- T cells had improved survival and decreased CD69 expression in response to PALNA compared to WT PALNA-treated cells. Conclusions: STING contributes to T cell inflammation in experimental cardiometabolic HFpEF by synergizing with dysregulated ER stress responses to promote the emergence of hyperactivated T cells.

Background Aging‐associated left ventricular dysfunction promotes cardiopulmonary fibrogenic remodeling, Group 2 pulmonary hypertension (PH), and right ventricular failure. At the time of diagnosis, cardiac function has declined, and cardiopulmonary fibrosis has often developed. Here, we sought to develop a molecular positron emission tomography (PET)–magnetic resonance imaging (MRI) protocol to detect both cardiopulmonary fibrosis and fibrotic disease activity in a left ventricular dysfunction model. Methods and Results Left ventricular dysfunction was induced by transverse aortic constriction (TAC) in 6‐month‐old senescence‐accelerated prone mice, a subset of mice that received sham surgery. Three weeks after surgery, mice underwent simultaneous PET‐MRI at 4.7 T. Collagen‐targeted PET and fibrogenesis magnetic resonance (MR) probes were intravenously administered. PET signal was computed as myocardium‐ or lung‐to‐muscle ratio. Percent signal intensity increase and Δ lung‐to‐muscle ratio were computed from the pre‐/postinjection magnetic resonance images. Elevated allysine in the heart ( P =0.02) and lungs ( P =0.17) of TAC mice corresponded to an increase in myocardial magnetic resonance imaging percent signal intensity increase ( P <0.0001) and Δlung‐to‐muscle ratio ( P <0.0001). Hydroxyproline in the heart ( P <0.0001) and lungs ( P <0.01) were elevated in TAC mice, which corresponded to an increase in heart (myocardium‐to‐muscle ratio, P =0.02) and lung (lung‐to‐muscle ratio, P <0.001) PET measurements. Pressure‐volume loop and echocardiography demonstrated adverse left ventricular remodeling, function, and increased right ventricular systolic pressure in TAC mice. Conclusions Administration of collagen‐targeted PET and allysine‐targeted MR probes led to elevated PET–magnetic resonance imaging signals in the myocardium and lungs of TAC mice. The study demonstrates the potential to detect fibrosis and fibrogenesis in cardiopulmonary disease through a dual molecular PET–magnetic resonance imaging protocol.

[This corrects the article DOI: 10.3389/fcvm.2023.1223244.].

Barth Syndrome (BTHS) is an early onset, lethal X-linked disorder caused by a mutation in tafazzin (TAFAZZIN), a mitochondrial acyltransferase that remodels monolysocardiolipin (MLCL) to mature cardiolipin (CL) and is essential for normal mitochondrial, cardiac, and skeletal muscle function. Current gene therapies in preclinical development require high levels of transduction. We tested whether TAFAZZIN gene therapy could be enhanced with the addition of a cell-penetrating peptide, penetratin (Antp). We found that TAFAZZIN-Antp was more effective than TAFAZZIN at preventing the development of pathological cardiac hypertrophy and heart failure. These findings indicate that a cell-penetrating peptide enhances gene therapy for BTHS.

Introduction: Cardiomyocyte (CM) hypertrophy, endothelial cell (EC) dysfunction and cardiac inflammation are hallmarks of adverse cardiac remodeling that contribute to heart failure (HF) progression. The “Stimulator of Interferon Genes” (STING) is highly expressed in ECs, the dominant non-CM cell in the heart and contributes to vascular inflammation, yet its role in adverse cardiac remodeling is unknown. We hypothesized that EC STING contributes to systolic dysfunction by modulating adverse remodeling in HF. Methods: Wildtype (WT), global deficient (STING-/-) and inducible EC STING-/- mice or EC STING+/+ littermate controls (Cad5ERTCre2+/–Stingfl/fl treated with Tamoxifen or oil, respectively) were subjected to transverse aortic constriction (TAC) or Sham surgery for 2-8 weeks. Cardiac function was analyzed by echocardiography. Left ventricular (LV) sections were stained with wheat germ agglutinin and isolectin to analyze CM hypertrophy and capillary density. Cardiac leukocytes were analyzed by flow cytometry and gene expression by qPCR. Bulk RNA Sequencing was performed on sorted CD45- CD31+ mouse heart ECs (MHEC) from EC STING+/+ and EC STING-/- after 4 weeks of TAC or sham surgery. ELISA was performed on MHEC supernatant. Results: STING-/- mice did not show a decline in fractional shortening (FS), capillary density or develop LV hypertrophy and had decreased intramyocardial CD45+ leukocytes in response to TAC, compared to WT mice. EC STING-/- mice also showed preserved FS, capillary density, and ameliorated CM hypertrophy over the course of 2, 4 and 8 weeks of TAC, compared to EC STING+/+ mice, despite having similar numbers of myocardial CD45+ leukocytes. RNASeq revealed decreased IL6 transcription, a pro-hypertrophic cytokine, in TAC STING-/- MHECs compared to WT MHECs. Myocardial IL6 gene expression was also reduced in EC STING-/- TAC hearts compared to EC STING+/+. In vitro, STING-/- MHECs had decreased secretion of IL6 protein compared to WT MHECs. Conclusions: Our data demonstrate that EC STING modulates both the EC and cardiac expression of the pro-hypertrophic cytokine IL6, suggesting a mechanism contributing to cardiomyocyte hypertrophy, capillary rarefaction, and systolic dysfunction in response to cardiac pressure overload.

Study Objective: To determine the cardiac myocyte (CM) and blood pressure-independent effects of Mixed lineage kinase 3 (MLK3) in the regulation of left ventricular (LV) function at baseline and in response to pathological stress. Background: MLK3 functions as a direct substrate effector of cGMP-dependent protein kinase 1α (PKG1α). Whole-body MLK3 deletion in mice increases cardiac dysfunction and remodeling after pressure overload and prevents the therapeutic effect of cGMP augmentation on LV function. However, MLK3 deletion also causes hypertension, which may confound these findings. Hypothesis: MLK3 opposes LV dysfunction through a blood pressure-independent function in the cardiac myocyte (CM). Methods: We generated mice harboring LoxP sites flanking the MAP3K11 gene encoding MLK3 (MLK3fl/fl) and crossed them with αMHC-Cre transgenic mice, enabling constitutive postnatal cardiac myocyte-specific MLK3 deletion (CMKO). Male and female 3- and 6-month-old MLK3 CMKO and control MLK3 intact (MLK3fl/fl Cre-) littermates were studied in the basal state. We also performed transaortic constriction (TAC) or sham surgery for 4 weeks on 3-month-old male mice. We assessed LV structure and function by organ mass, echocardiography, pressure volume loop analysis, qPCR, and immunoblot. In basal and TAC studies, we used age-matched αMHC-Cre x MLK3+/+ (non-floxed) mice as additional controls. Results: MLK3 gene excision in CMs was confirmed by PCR. By 6 months of age, MLK3 CMKO mice displayed: progressive reduction in LV ejection fraction; increase in LV internal diameter; and LV hypertrophy, indicating LV dysfunction and pathological remodeling, compared with littermate controls or with αMHC-Cre x MLK3+/+ non-floxed mice. This effect was more prominent in females than in males. LV fetal gene expression did not differ between genotypes at 3 months. At 6 months of age, however, the fetal genes nppa, RCAN, and CTGF were significantly upregulated in both male and female MLK3 CMKO mice, compared with MLK3 intact, indicating pathological remodeling. After 4-week TAC, MLK3 CMKO LVs developed more severe reduction in LV systolic function compared with MLK3 intact littermates as assayed by echocardiographic measures of LV ejection fraction; and invasive hemodynamic indices of preload recruitable stroke work, maximum LV pressure and LV developed pressure. The MLK3 CMKO LVs also demonstrated a more eccentric remodeling phenotype in which LV mass/tibia length, LV end diastolic and end systolic diameters increased more severely in CMKO mice, with corresponding reduced LV wall thicknesses. Finally, lung mass/tibia length was elevated in the MLK3 CMKO TAC mice compared to MLK3 intact TAC littermates, indicating overt heart failure. Conclusion: (1) MLK3 functions as a tonic inhibitor of LV dysfunction and pathological remodeling through a role in the CM, possibly through sex-specific mechanisms. (2) Intact MLK3 is required for normal LV compensation and concentric remodeling in response to pressure overload. These findings have important clinical implications, as drugs which activate PKG1 in heart failure improve outcomes but are limited by excess hypotension. As a myocardial PKG1 substrate which opposes pathological LV remodeling through blood pressure-independent mechanisms, MLK3 may represent a candidate therapeutic target for heart failure. NIH 1R01HL162919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heart failure (HF) is highly prevalent. Mechanisms underlying HF remain incompletely understood. Splicing factors (SF), which control pre-mRNA alternative splicing, regulate cardiac structure and function. This study investigated regulation of the splicing factor heterogeneous nuclear ribonucleoprotein-L (hnRNPL) in the failing heart. hnRNPL protein increased in left ventricular tissue from mice with transaortic constriction-induced HF and from HF patients. In left ventricular tissue, hnRNPL was detected predominantly in nuclei. Knockdown of the hnRNPL homolog Smooth in Drosophila induced cardiomyopathy. Computational analysis of predicted mouse and human hnRNPL binding sites suggested hnRNPL-mediated alternative splicing of tropomyosin, which was confirmed in C2C12 myoblasts. These findings identify hnRNPL as a sensor of cardiac dysfunction and suggest that disturbances of hnRNPL affect alternative splicing in HF.

Introduction: Mixed-lineage kinase 3 (MLK3) is a cGMP-dependent protein kinase 1α (PKG1α) substrate, preserving left ventricular (LV) function without affecting blood pressure (BP). MLK3 kinase-dependent effects maintain LV function, while kinase-independent signalling regulates BP. Thus, enhancing MLK3 kinase activity may improve LV function in heart failure (HF) while circumventing PKG1-induced hypotension. No MLK3-activation strategies exist currently, thus hindering progress in MLK3 research and potential therapeutics. Hypothesis: Augmenting MLK3 after pressure overload by transaortic constriction (TAC) would prevent LV hypertrophy and dysfunction in mice. Goal: To test the effect of adeno-associated virus 9 (AAV9)-mediated MLK3 overexpression on LV function, when administered in the setting of pre-existing LV hypertrophy and dysfunction. Methods: Male mice 12 weeks of age underwent 26G TAC or sham surgery. At day 5 post-TAC we administered by retro-orbital injection AAV9 encoding MLK3 or TdTomato negative control, each under control of the CM-specific cTNT promoter (AAV-MLK3: n=12 TAC, 7 sham; AAV-TdTomato: n=10 Tac, 8 sham. At day 28 post-AAV9 injection, we measured cardiac structure and function by echocardiography, invasive LV hemodynamics, and organ weights. Results: Echocardiography 5 days post-TAC confirmed TAC-induced LV hypertrophy and reduced systolic function prior to AAV9 treatment. At day 28, AAV-MLK3-treated TAC mice demonstrated improved LV ejection fraction and fractional shortening compared with TAC-AAV-Td controls, indicating improved systolic function. MLK3-treated TAC mice maintained higher LV systolic pressure post-TAC vs. Td controls, further indicating improved cardiac performance. Diastolic indices, including ratio of mitral E/A inflow velocities, tau, and LV relaxation rate were also improved in AAV-MLK3 TAC versus AAV-Td controls. AAV9-MLK3 administration had no effect on LV wall thickness or on LV or whole heart mass normalized to tibia length. MLK3 expression was increased 3-fold in AAV9-MLK3 TAC LVs compared with AAV9-Td controls. Conclusion: AAV9-mediated MLK3 overexpression after the onset of LV hypertrophy and dysfunction improves LV function in mice.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation. While mutations in sarcomere genes can account for a substantial proportion of familial cases, 40-50% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYAB R123W ) in a pair of homozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB R123W promotes HCM phenotype remains unclear. Here, we generated mice carrying the Cryab R123W -knockin allele and demonstrated that hearts from these animals exhibit increased maximal elastance, reduced diastolic function and are more susceptible to ventricular tachycardia with programmed stimulation. Upon transverse aortic constriction, mice carrying the Cryab R123W allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased ejection fraction. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB R123W despite its potent effect on driving cellular hypertrophy. Unexpectedly, CRYAB R123W appears to enhance calcium signaling by promoting nuclear localization of NFAT through direct interaction with calcineurin. Studies on isolated adult cardiomyocytes reveal altered fractional shortening, calcium dyshomeostasis, prolonged action potential duration, and T-wave alternans. Crossing of CRYAB R123W mice with a mybpc3 knock-in model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in this model are independent of the sarcomere. Thus, our data establish the Cryab R123W allele as a novel genetic model of HCM that potentially unveils additional sarcomere-independent mechanisms of cardiac pathological hypertrophy and sudden cardiac death.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in sarcomere genes can account for a substantial proportion of familial cases of HCM, 40%–50% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYABR123W) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYABR123W promotes the HCM phenotype remains unclear. Here, we generated mice carrying the CryabR123W knock-in allele and demonstrated that hearts from these animals exhibit increased maximal elastance at young age but reduced diastolic function with aging. Upon transverse aortic constriction, mice carrying the CryabR123W allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased ejection fraction. Crossing of mice with a Mybpc3 frame-shift model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in the CryabR123W model are independent of the sarcomere. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYABR123W despite its potent effect on driving cellular hypertrophy. Mechanistically, we uncovered an unexpected protein-protein interaction between CRYAB and calcineurin. Whereas CRYAB suppresses maladaptive calcium signaling in response to pressure-overload, the R123W mutation abolished this effect and instead drove pathologic NFAT activation. Thus, our data establish the CryabR123W allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiac pathological hypertrophy.