ABSTRACT Background Cardiomyocytes respond to stress by undergoing hypertrophic growth driven by dynamic changes in gene expression. Epigenetic mechanisms, including histone methylation, play critical roles in regulating these transcriptional programs, yet the enzymes controlling these modifications during cardiac disease remain largely unknown. The SMYD family of histone methyltransferases regulates gene expression in multiple biological contexts, but the function of SMYD5 in the mammalian heart has never been investigated. Methods SMYD5 expression was assessed in human heart failure samples and in a mouse model of cardiac hypertrophy. To define its functional role in vivo, we generated inducible cardiomyocyte-specific Smyd5 knockout mice and characterized their cardiac phenotype using molecular, histological, and functional analyses. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine histone H4 lysine 20 trimethylation (H4K20me3) at the Il-6 promoter. Results SMYD5 expression was altered in diseased human and mouse hearts. Under basal conditions, cardiomyocyte-specific deletion of Smyd5 resulted in baseline structural cardiac remodeling and transcriptional signatures characteristic of pathological stress. Smyd5 -deficient hearts exhibited marked inflammatory activation resembling a cytokine storm with immune cell infiltration and heart failure. Notably, Smyd5 knockout mice displayed a 100-fold increase in Il-6 expression, accompanied by a global reduction in H4K20me3. ChIP-qPCR analysis of the Il-6 promoter, together with loss- and gain-of-function analysis of SMYD5, supports a direct epigenetic role of SMYD5 in regulating Il-6 expression through H4K20me3 in cardiomyocytes. Conclusions SMYD5 is a previously unrecognized epigenetic regulator of cardiac homeostasis that restrains inflammatory signaling in cardiomyocytes under normal conditions. Loss of Smyd5 disrupts H4K20me3, leading to derepression of Il-6 in cardiomyocytes and a robust inflammatory response characterized by immune cell recruitment and fibrosis, accompanied by rapid progression of cardiac remodeling and heart failure. These findings identify SMYD5 as a critical regulator of intrinsic cardiomyocyte inflammatory signaling and reveal a novel chromatin-based mechanism contributing to inflammatory cardiomyopathies. NOVELTY AND SIGNIFICANCE What Is Known? Elevated levels of pro-inflammatory cytokines such as IL-6 are strongly associated with adverse cardiac remodeling and poor outcomes in heart failure. Histone methylation is a key epigenetic mechanism regulating gene expression in the heart, but the role of histone H4K20 trimethylation and its regulatory enzymes in cardiomyocytes remains poorly understood. The histone methyltransferase SMYD5 regulates gene expression and inflammatory pathways in several non-cardiac cell types, but its role in the mammalian heart has not been examined. What New Information Does This Article Contribute? This study provides the first in vivo characterization of SMYD5 in the heart and identifies it as a critical epigenetic regulator of cardiomyocyte homeostasis. Cardiomyocyte-specific loss of Smyd5 triggers pathological hypertrophy, elevated inflammatory signaling, immune cell infiltration, fibrosis, and heart failure. SMYD5 directly represses Il-6 expression in cardiomyocytes through histone H4K20 trimethylation at the Il-6 promoter, revealing a previously unrecognized epigenetic mechanism controlling intrinsic cardiomyocyte-driven inflammation. Epigenetic mechanisms that regulate inflammatory signaling in cardiomyocytes remain largely unknown. Here, we identify the histone methyltransferase SMYD5 as a critical regulator of cardiac homeostasis and intrinsic inflammatory signaling. Using an inducible cardiomyocyte-specific Smyd5 knockout mouse model, we demonstrate that loss of Smyd5 induces rapid progression to heart failure accompanied by robust inflammatory activation, including a ∼100-fold increase in Il-6 expression, inflammatory immune cell infiltration, fibrosis, and severe cardiac dysfunction. Mechanistically, SMYD5 directly regulates Il-6 expression by catalyzing histone H4 lysine 20 trimethylation at the Il-6 promoter, thereby restraining pro-inflammatory gene expression in cardiomyocytes. Ablation of Smyd5 markedly reduces global H4K20 trimethylation, and results in dramatic upregulation of Il-6 and downstream cytokine signaling pathways, producing a phenotype resembling cytokine storm-like inflammatory cardiomyopathy. These findings establish SMYD5 as the first epigenetic regulator shown to suppress intrinsic cardiomyocyte inflammatory signaling and uncover a novel chromatin-based mechanism controlling cytokine production in the heart. Targeting SMYD5-dependent pathways therefore represents a new strategy for limiting maladaptive inflammation in heart failure and inflammatory cardiomyopathies.

Histone H4K20 methylation is critical in regulating the cell cycle, DNA damage response, and gene repression in proliferating cells. However, its role in the heart remains poorly understood. Our previous work revealed that histone H4K20 tri-methylation is elevated in acute cell models of cardiomyocyte hypertrophy but is reduced in mouse models of cardiac hypertrophy and ischemia. Although these findings highlight the dynamic nature of this modification and its significance in regulating gene expression, the data on enzymes regulating H4K20 methylation is sparse. To build upon this work and investigate H4K20 di-methylation and the enzymes modulating this site in cardiac pathology, we quantified histone H4K20 di-methylation and 12 methyltransferases and demethylases across one cell model, two mouse models of cardiac dysfunction, and cardiac tissue from heart failure patients. While we observed no global changes in H4K20 di-methylation, we detected alterations in methyltransferases KMT5C and SMYD5 and demethylases RAD23A and KDM7C in humans and mice. These findings suggest changes in H4K20 di-methylation may occur on an individual gene basis but do not lead to global alterations in H4K20 di-methylation. Additionally, this work identified four enzymes differentially modulated in cardiac dysfunction to advance our understanding of epigenetic mechanisms involved in heart disease.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

SMYD1 is a lysine methyltransferase, which has been shown to methylate lysine 4 on histone H3, an established mark of gene activation. SMYD1 is only expressed in skeletal and cardiac muscle and was originally shown to play a significant role in regulating cardiac development. In the adult myocardium, using inducible, cardiomyocyte-specific Smyd1 knockout mice, loss of SMYD1 leads to massive downregulation of mitochondrial bioenergetics and overt heart failure. However, the effects of SMYD1 gain-of-function in the heart and its molecular function in the cardiomyocyte in response to ischemic stress remains unknown. Here we demonstrate that SMYD1a, the mouse ortholog of human SMYD1, positively regulates cardiac energetics and protects the heart from ischemic injury. To delineate how SMYD1a controls energy efficiency and metabolism in the cardiomyocyte, we generated a novel mouse model capable of inducible cardiomyocyte-specific SMYD1a overexpression. When subjected to ischemic injury these transgenic mice display reduced infarct size and cardiomyocyte death concomitant with enhanced mitochondrial respiratory efficiency. In addition, our molecular analysis revealed that the cardiac tissue in these animals is protected from ischemic injury through SMYD1a’s synergistic regulation of two key mitochondrial pathways. First, through its histone methyltransferase activity, SMYD1a maintains metabolic homeostasis by preserving basal expression of PGC-1α and its downstream targets including electron transport chain subunits. Second, SMYD1a regulates expression of OPA1, a key regulator of cristae morphology which drives the formation of electron transport chain supercomplexes to enhance mitochondrial respiration and ATP production. This work highlights SMYD1a as the only known epigenetic regulator of cristae morphology and identifies a novel molecular pathway by which cardiomyocytes dynamically regulate energy efficiency to protect from ischemic injury.

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to >50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

SMYD1a, a myosin-specific histone lysine methyltransferase, plays a major role in regulating disease-induced remodeling in the adult heart. Previously, we demonstrated that the inducible loss of this chromatin-bound enzyme is sufficient to induce cardiac hypertrophy and failure in vivo , which is preceded by downregulation of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS), and reduction of mitochondrial respiration capacity. However, our most recent data in transgenic mice (TG) displaying inducible, cardiomyocyte-specific overexpression of SMYD1a show that these mice are protected from ischemic injury after permanent occlusion (PO) of the LAD manifested by reduced infarct size and cardiac dysfunction compared to littermate controls (WT), suggesting that SMYD1 plays a protective role in the heart and mitigates disease-induced remodeling. Additionally, global proteomic evaluation of cardiac tissue from TG mice showed unique expression of metabolic enzymes, including proteins from the electron transport chain, and our high-resolution mitochondrial respirometry analysis showed that overexpression of SMYD1a leads to increased oxygen consumption rates through Complex I and II. To further asses OXPHOS efficiency in TG mice we subjected them to permanent occlusion of the LAD and evaluated ATP production rates in isolated mitochondria from TG and WT mice, by measuring the molar amount of ATP produced per mole of atomic oxygen consumed (known as ATP:O ratio). Interestingly, we observed a significant increase in ATP:O ratio in TG mice 24h after PO suggesting that they are much more efficient at producing ATP. Finally, we show that the global regulation of mitochondrial respiration in TG mice occurs through transcriptional control of Ppargc1α . Our results confirm that cardiac expression of Ppargc1α was significantly reduced in WT mice (48h after PO) but maintained at basal levels in TG mice, which also corroborated with our ChIP-qPCR data showing SMYD1a binding to the Ppargc1α promoter and regulating its expression. Overall, these results show that SMYD1a can mitigate ischemic injury and adverse remodeling in the adult myocardium, which occurs through Ppargc1α expression and regulation of cardiac energetics and metabolism.

Protein methylation plays a pivotal role in the regulation of various cellular processes including chromatin remodeling and gene expression. SET and MYND domain-containing proteins (Smyd) are a special class of lysine methyltransferases whose catalytic SET domain is split by an MYND domain. The hallmark feature of this family was thought to be the methylation of histone H3 (on lysine 4). However, several studies suggest that the role of the Smyd family is dynamic, targeting unique histone residues associated with both transcriptional activation and repression. Smyd proteins also methylate several non-histone proteins to regulate various cellular processes. Although we are only beginning to understand their specific molecular functions and role in chromatin remodeling, recent studies have advanced our understanding of this relatively uncharacterized family, highlighting their involvement in development, cell growth and differentiation and during disease in various animal models. This review summarizes our current knowledge of the structure, function and methylation targets of the Smyd family and provides a compilation of data emphasizing their prominent role in cardiac and skeletal muscle physiology and pathology.

Heart disease is the leading cause of death in the United States. This chronic condition affects the heart so it can no longer sufficiently pump blood to vital organs in the body. While heart disease can manifest in many different ways the general pathophysiology is conserved: the heart compensates for an increased workload by undergoing hypertrophic growth, accompanied by transcriptional reprogramming, and progresses into heart failure. Although many epigenetic factors have been identified which influence these underlying changes in transcription during disease, we still know very little about how this process is regulated in the cardiomyocyte. Smyd1, a unique histone methyltransferase that regulates gene expression in the cardiomyocyte, was originally shown to play a crucial role in early cardiac development. More recently, we have shown that loss of Smyd1 in the adult mouse heart leads to pro-hypertrophic signaling resulting in cardiomyocyte growth, fibrosis and functional decline. In addition, we examined the two Smyd1 isoforms (Smyd1a and Smy1b) in isolated myocytes and showed that Smyd1a (but not Smyd1b) overexpression (OE) was capable of inhibiting phenylephrine-induced hypertrophy. To further characterize the role of Smyd1 in the myocardium and determine if Smyd1a is capable of inhibiting hypertrophic growth in adult heart we generated transgenic mice capable of inducible, cardiac-specific OE of Smyd1a using a Tet-On system. Mice were fed doxycycline-laced chow for 2 weeks to achieve Smyd1a-OE at which time we characterized their cardiac phenotype under basal conditions and after stress (permanent occlusion). Interestingly, transgenic mice showed no observable difference under basal conditions, however, 3 weeks after permanent occlusion of the LAD, the EF of wild type animals decreased to ~37%, while transgenic mice maintained an EF of ~61% . In addition, transgenic mice exhibited exacerbated growth (12% increase in HW/BW ratio) with no change in expression of the hypertrophic marker ANF, in contrast to wild type animals (4 fold increase). These exciting results suggest that Smyd1a is capable of preserving cardiac function in adult mice, however, this mechanism appears to be independent of hypertrophic signaling.

Heart disease is the most financially debilitating and fatal disease between both men and women in the United States. Heart failure is defined as the point at which the heart's ability to function declines, due to overexertion, and can no longer sufficiently pump blood to vital organs throughout the body. While there are different forms of heart disease the pathophysiology of these is largely conserved: the heart compensates for an increased work load by undergoing hypertrophic growth and ultimately progresses onto heart failure. The Smyd family of histone methyltransferases has been studied for their involvement in cell growth during development and disease and is thought to accomplish this by regulating gene expression through histone methylation. While the roles of Smyd1, 2, and 3 are beginning to be defined, the remaining two family members, Smyd4 and 5, are virtually uncharacterized. My hypothesis is that Smyd5 is involved in inhibiting hypertrophic growth in the myocardium, however, its role in the heart is completely unknown. To determine the function of Smyd5, we generated constitutive and inducible, cardiac‐specific Smyd5 knockout mice and have characterized their cardiac phenotype under basal conditions and after stress (pressure‐overload hypertrophy and isoproterenol infusion). When compared to wild type mice, these animals display elevated heart weight/body ratios and left ventricular wall thickening as well as increased fibrosis and changes in fetal gene expression normally detected in cardiac hypertrophy. In addition knockout of Smyd5 significantly reduces methylation of lysine 20 on histone H4, suggesting a mechanistic basis for this phenotype. Together, our results show that although Smyd5 is dispensable for cardiac development, it is a critical regulator of hypertrophic cell growth and pathologic gene expression in the cardiomyocyte and highlights a novel role for this histone methyltransferase in the myocardium.Support or Funding InformationACCESS, BioURP, APS (UGREF), UROP, CVRTI

While global changes in gene expression are a hallmark of heart disease, much less is known regarding the epigenetic factors driving these changes. Local chromatin packing and gene accessibility, which governs transcriptional status, has been correlated with specific post‐translational modifications (PTMs) on the histone tails of nucleosomes occupying these regions. However, the specific alterations in histone PTMs driving the conserved gene expression changes observed in heart disease and how these ultimately affect cardiac physiology are largely unknown. To identify changes in histone PTMs during disease progression, we performed label‐free quantitation of acid‐extracted proteins from mouse heart (under basal, hypertrophic, and failing conditions) and from isolated ventricular myocytes exposed to hypertrophic agonists, a cell model of cardiac hypertrophy. Protein samples were propionylated, enzymatically digested and analyzed on an Orbitrap Velos mass spectrometer and differential expression analysis was performed using MaxQuant and Perseus software. Collectively we identified 55 and 30 PTMs spanning the five histone families, from cardiac tissue and isolated myocytes, respectively, 16 of which were differentially regulated in the setting of disease. Additionally our analysis of large peptides containing multiple PTMs enabled us to determine how these modifications are regulated with respect to one another providing insights into the broader histone code. This study presents the first global characterization of histone post‐translational modifications in the heart and highlights basic mechanisms of genomic reprogramming operative in disease.