YTHDF1 is pivotal for maintenance of cardiac homeostasis.

The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.

Loss of YTHDF2 Alters the Expression of m6A-Modified Myzap and Causes Adverse Cardiac Remodeling.

How post-transcriptional regulation of gene expression, such as through N6-methyladenosine (m6A) messenger RNA methylation, impacts heart function is not well understood. We found that loss of the m6A binding protein YTHDF2 in cardiomyocytes of adult mice drove cardiac dysfunction. By proteomics, we found myocardial zonula adherens protein (MYZAP) within the top up-regulated proteins in knockout cardiomyocytes. We further demonstrated that YTHDF2 binds m6A-modified Myzap messenger RNA and controls its stability. Cardiac overexpression of MYZAP has been associated with cardiomyopathy. Thus, our findings provide an important new mechanism for the YTHDF2-dependent regulation of this target and therein its novel role in the maintenance of cardiac homeostasis.

Micro-dystrophin gene therapy demonstrates long-term cardiac efficacy in a severe Duchenne muscular dystrophy model.

Micro-dystrophin gene replacement therapies for Duchenne muscular dystrophy (DMD) are currently in clinical trials, but have not been thoroughly investigated for their efficacy on cardiomyopathy progression to heart failure. We previously validated Fiona/dystrophin-utrophin-deficient (dko) mice as a DMD cardiomyopathy model that progresses to reduced ejection fraction indicative of heart failure. Adeno-associated viral (AAV) vector delivery of an early generation micro-dystrophin prevented cardiac pathology and functional decline through 1 year of age in this new model. We now show that gene therapy using a micro-dystrophin optimized for skeletal muscle efficacy (AAV-µDys5), and which is currently in a clinical trial, is able to fully prevent cardiac pathology and cardiac strain abnormalities and maintain normal (>45%) ejection fraction through 18 months of age in Fiona/dko mice. Early treatment with AAV-µDys5 prevents inflammation and fibrosis in Fiona/dko hearts. Collagen in cardiac fibrotic scars becomes more tightly packed from 12 to 18 months in Fiona/dko mice, but the area of fibrosis containing tenascin C does not change. Increased tight collagen correlates with unexpected improvements in Fiona/dko whole-heart function that maintain impaired cardiac strain and strain rate. This study supports micro-dystrophin gene therapy as a promising intervention for preventing DMD cardiomyopathy progression.

Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis.

Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe.

BEX1 is a critical determinant of viral myocarditis.

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-ß) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.

The m6A methyltransferase METTL3 regulates muscle maintenance and growth in mice.

Skeletal muscle serves fundamental roles in organismal health. Gene expression fluctuations are critical for muscle homeostasis and the response to environmental insults. Yet, little is known about post-transcriptional mechanisms regulating such fluctuations while impacting muscle proteome. Here we report genome-wide analysis of mRNA methyladenosine (m6A) dynamics of skeletal muscle hypertrophic growth following overload-induced stress. We show that increases in METTL3 (the m6A enzyme), and concomitantly m6A, control skeletal muscle size during hypertrophy; exogenous delivery of METTL3 induces skeletal muscle growth, even without external triggers. We also show that METTL3 represses activin type 2 A receptors (ACVR2A) synthesis, blunting activation of anti-hypertrophic signaling. Notably, myofiber-specific conditional genetic deletion of METTL3 caused spontaneous muscle wasting over time and abrogated overload-induced hypertrophy; a phenotype reverted by co-administration of a myostatin inhibitor. These studies identify a previously unrecognized post-transcriptional mechanism promoting the hypertrophic response of skeletal muscle via control of myostatin signaling.

Cardiac-derived TGF-ß1 confers resistance to diet-induced obesity through the regulation of adipocyte size and function.

Regulation of organismal homeostasis in response to nutrient availability is a vital physiological process that involves inter-organ communication. Understanding the mechanisms controlling systemic cross-talk for the maintenance of metabolic health is critical to counteract diet-induced obesity. Here, we show that cardiac-derived transforming growth factor beta 1 (TGF-ß1) protects against weight gain and glucose intolerance in mice subjected to high-fat diet. Secretion of TGF-ß1 by cardiomyocytes correlates with the bioavailability of this factor in circulation. TGF-ß1 prevents adipose tissue inflammation independent of body mass and glucose metabolism phenotypes, indicating protection from adipocyte dysfunction-driven immune cell recruitment. TGF-ß1 alters the gene expression programs in white adipocytes, favoring their fatty acid oxidation and consequently increasing their mitochondrial oxygen consumption rates. Ultimately, subcutaneous and visceral white adipose tissue from cadiac-specific TGF-ß1 transgenic mice fail to undergo cellular hypertrophy, leading to reduced overall adiposity during high-fat feeding. Thus, TGF-ß1 is a critical mediator of heart-fat communication for the regulation of systemic metabolism.

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure.

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.

Micro-dystrophin gene therapy prevents heart failure in an improved Duchenne muscular dystrophy cardiomyopathy mouse model.

Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy in preventing heart failure is unknown. Although most patients with DMD die from heart failure, cardiomyopathy is undetectable until the teens, so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure, and dog models showed highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model to our knowledge that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevented declines in cardiac function and prohibited onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and nongenetic therapies to optimize cardiac care for patients with DMD.

Microfibrillar-Associated Protein 4 Regulates Stress-Induced Cardiac Remodeling.

[Figure: see text].

Paracardial fat remodeling affects systemic metabolism through alcohol dehydrogenase 1.

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.

Remodeling of the m6A landscape in the heart reveals few conserved post-transcriptional events underlying cardiomyocyte hypertrophy.

Regulation of gene expression plays a fundamental role in cardiac stress-responses. Modification of coding transcripts by adenosine methylation (m6A) has recently emerged as a critical post-transcriptional mechanism underlying heart disease. Thousands of mammalian mRNAs are known to be m6A-modified, suggesting that remodeling of the m6A landscape may play an important role in cardiac pathophysiology. Here we found an increase in m6A content in human heart failure samples. We then adopted genome-wide analysis to define all m6A-regulated sites in human failing compared to non-failing hearts and identified targeted transcripts involved in histone modification as enriched in heart failure. Further, we compared all m6A sites regulated in human hearts with the ones occurring in isolated rat hypertrophic cardiomyocytes to define cardiomyocyte-specific m6A events conserved across species. Our results identified 38 shared transcripts targeted by m6A during stress conditions, and 11 events that are unique to unstressed cardiomyocytes. Of these, further evaluation of select mRNA and protein abundances demonstrates the potential impact of m6A on post-transcriptional regulation of gene expression in the heart.

IFITM3 protects the heart during influenza virus infection.

Influenza virus can disseminate from the lungs to the heart in severe infections and can induce cardiac pathology, but this has been difficult to study due to a lack of small animal models. In humans, polymorphisms in the gene encoding the antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) are associated with susceptibility to severe influenza, but whether IFITM3 deficiencies contribute to cardiac dysfunction during infection is unclear. We show that IFITM3 deficiency in a new knockout (KO) mouse model increases weight loss and mortality following influenza virus infections. We investigated this enhanced pathogenesis with the A/PR/8/34 (H1N1) (PR8) influenza virus strain, which is lethal in KO mice even at low doses, and observed increased replication of virus in the lungs, spleens, and hearts of KO mice compared with wild-type (WT) mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, including decreased heart rate and irregular, arrhythmic RR (interbeat) intervals, whereas WT mice exhibited a mild decrease in heart rate without irregular RR intervals. Cardiac electrical dysfunction in PR8-infected KO mice was accompanied by increased activation of fibrotic pathways and fibrotic lesions in the heart. Infection with a sublethal dose of a less virulent influenza virus strain (A/WSN/33 [H1N1]) resulted in a milder cardiac electrical dysfunction in KO mice that subsided as the mice recovered. Our findings reveal an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and establish IFITM3 KO mice as a powerful model for studying mild and severe influenza virus-induced cardiac dysfunction.

RNA epigenetics and cardiovascular diseases.

Cardiovascular disease (CVD) remains the leading cause of death in the Western world. Despite advances in the prevention and in the management of CVD, the role of RNA epigenetics in the cardiovascular system has been until recently unexplored. The rapidly expanding research field of RNA modifications has introduced a novel layer of gene regulation in mammalian cells. RNA modifications may control all aspects of RNA metabolism, and their study reveals previously unrecognized regulatory pathways that may determine gene expression at a post-transcriptional level. Understanding the role of RNA modifications in CVD may lead towards a better understanding of disease mechanisms and the development of novel biomarkers or therapeutic strategies. In this review, we highlight the most recent and major reports in the field of RNA methylation and adenosine to inosine RNA editing related to the cardiovascular field and we discuss how this breakthrough will advance the field of precision medicine.

The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy.

BACKGROUND: N6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. METHODS: To determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. RESULTS: We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis. CONCLUSIONS: Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.

CTGF/CCN2 is an autocrine regulator of cardiac fibrosis.

Cardiac fibrosis is a common pathologic consequence of stress insult to the heart and is characterized by abnormal deposition of fibrotic extracellular matrix that compromises cardiac function. Cardiac fibroblasts are key mediators of fibrotic remodeling and are regulated by secreted stress-response proteins. The matricellular protein connective tissue growth factor (CTGF), or CCN2, is strongly produced by injured cardiomyocytes and although it is considered a pro-fibrotic factor in many organ systems, its role in cardiac fibrosis is controversial. Here we adopted a cell-specific genetic approach to conditionally delete CCN2 in either cardiomyocytes or activated fibroblasts. Fibrosis was induced by angiotensin II-based neurohumoral stimulation, an insult that strongly induces CCN2 expression from cardiomyocytes and to a lesser extent in fibroblasts. Remarkably, only CCN2 deletion from activated fibroblasts inhibited the fibrotic remodeling while deletion from cardiomyocytes (the main source of CCN2 in the heart) had no effects. In vitro experiments revealed that although efficiently secreted by both fibroblasts and cardiomyocytes, only fibroblast-derived CCN2 is proficient in its ability to fully activate fibroblasts. These results overall indicate that although secreted into the extracellular matrix, CCN2 acts in an autocrine fashion. Secretion of CCN2 by cardiomyocytes is not pro-fibrotic, while fibroblast-derived CCN2 can modulate fibrosis in the heart. In conclusion we found that cardiomyocyte-derived CCN2 is dispensable for cardiac fibrosis, while inhibiting CCN2 induction in activated fibroblasts is sufficient to abrogate the cardiac fibrotic response to angiotensin II. Hence, CCN2 is an autocrine factor in the heart.

At the heart of inter- and intracellular signaling: the intercalated disc.

Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.

Fibroblast-Specific Genetic Manipulation of p38 Mitogen-Activated Protein Kinase In Vivo Reveals Its Central Regulatory Role in Fibrosis.

BACKGROUND: In the heart, acute injury induces a fibrotic healing response that generates collagen-rich scarring that is at first protective but if inappropriately sustained can worsen heart disease. The fibrotic process is initiated by cytokines, neuroendocrine effectors, and mechanical strain that promote resident fibroblast differentiation into contractile and extracellular matrix-producing myofibroblasts. The mitogen-activated protein kinase p38α (Mapk14 gene) is known to influence the cardiac injury response, but its direct role in orchestrating programmed fibroblast differentiation and fibrosis in vivo is unknown. METHODS: A conditional Mapk14 allele was used to delete the p38α encoding gene specifically in cardiac fibroblasts or myofibroblasts with 2 different tamoxifen-inducible Cre recombinase-expressing gene-targeted mouse lines. Mice were subjected to ischemic injury or chronic neurohumoral stimulation and monitored for survival, cardiac function, and fibrotic remodeling. Antithetically, mice with fibroblast-specific transgenic overexpression of activated mitogen-activated protein kinase kinase 6, a direct inducer of p38, were generated to investigate whether this pathway can directly drive myofibroblast formation and the cardiac fibrotic response. RESULTS: In mice, loss of Mapk14 blocked cardiac fibroblast differentiation into myofibroblasts and ensuing fibrosis in response to ischemic injury or chronic neurohumoral stimulation. A similar inhibition of myofibroblast formation and healing was also observed in a dermal wounding model with deletion of Mapk14. Transgenic mice with fibroblast-specific activation of mitogen-activated protein kinase kinase 6-p38 developed interstitial and perivascular fibrosis in the heart, lung, and kidney as a result of enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum response factor and the signaling effector calcineurin. CONCLUSIONS: These findings suggest that signals from diverse modes of injury converge on p38α mitogen-activated protein kinase within the fibroblast to program the fibrotic response and myofibroblast formation in vivo, suggesting a novel therapeutic approach with p38 inhibitors for future clinical application.

Dysfunction of the ß2-spectrin-based pathway in human heart failure.

ß2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of ß2-spectrin for vertebrate function is illustrated by dysfunction of ß2-spectrin-based pathways in disease. Recently, defects in ß2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of ß2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that ß2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, ß2-spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Furthermore, compared with left ventricular samples from nonfailing hearts, ß2-spectrin levels were significantly decreased in left ventricle of ischemic- and nonischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced ß2-spectrin protein levels. Mechanistically, we identify that ß2-spectrin levels are tightly regulated by posttranslational mechanisms, namely Ca(2+)- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- and calpain-dependent loss of ß2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that ß2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis.