Big tau aggregation disrupts microtubule tyrosination and causes myocardial diastolic dysfunction: from discovery to therapy.

BACKGROUND: Amyloid plaques and neurofibrillary tangles, the molecular lesions that characterize Alzheimer's disease (AD) and other forms of dementia, are emerging as determinants of proteinopathies 'beyond the brain'. This study aims to establish tau's putative pathophysiological mechanistic roles and potential future therapeutic targeting of tau in heart failure (HF). METHODS AND RESULTS: A mouse model of tauopathy and human myocardial and brain tissue from patients with HF, AD, and controls was employed in this study. Tau protein expression was examined together with its distribution, and in vitro tau-related pathophysiological mechanisms were identified using a variety of biochemical, imaging, and functional approaches. A novel tau-targeting immunotherapy was tested to explore tau-targeted therapeutic potential in HF. Tau is expressed in normal and diseased human hearts, in contradistinction to the current oft-cited observation that tau is expressed specifically in the brain. Notably, the main cardiac isoform is high-molecular-weight (HMW) tau (also known as big tau), and hyperphosphorylated tau segregates in aggregates in HF and AD hearts. As previously described for amyloid-beta, the tauopathy phenotype in human myocardium is of diastolic dysfunction. Perturbation in the tubulin code, specifically a loss of tyrosinated microtubules, emerged as a potential mechanism of myocardial tauopathy. Monoclonal anti-tau antibody therapy improved myocardial function and clearance of toxic aggregates in mice, supporting tau as a potential target for novel HF immunotherapy. CONCLUSION: The study presents new mechanistic evidence and potential treatment for the brain-heart tauopathy axis in myocardial and brain degenerative diseases and ageing.

Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart.

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.

Is heart failure with preserved ejection fraction a 'dementia' of the heart?

Heart failure with preserved ejection fraction (HFpEF) remains an elusive entity, due to its heterogeneous clinical profile and an arbitrarily defined nosology. Several pathophysiological mechanisms recognized as central for the development of HFpEF appear to be in common with the process of physiological aging of the heart. Both conditions are characterized by progressive impairment in cardiac function, accompanied by left ventricular hypertrophy, diastolic dysfunction, sarcomeric, and metabolic abnormalities. The neurological paradigm of dementia-intended as a progressive, multifactorial organ damage with decline of functional reserve, eventually leading to irreversible dysfunction-is well suited to represent HFpEF. In such perspective, certain phenotypes of HFpEF may be viewed as a maladaptive response to environmental modifiers, causing premature and pathological aging of the heart. We here propose that the 'HFpEF syndrome' may reflect the interplay of adverse structural remodelling and erosion of functional reserve, mirroring the processes leading to dementia in the brain. The resulting conceptual framework may help advance our understanding of HFpEF and unravel potential therapeutical targets.

The long and winding road to target protein misfolding in cardiovascular diseases.

BACKGROUND: In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches have been introduced in the past, the development of novel treatments remains an important research goal, which is hampered by the lack of understanding of key mechanisms and targets. Emerging evidences in recent years indicate the involvement of misfolded proteins aggregation and the derailment of protein quality control in the pathogenesis of cardiovascular diseases. Several potential interventions targeting protein quality control have been translated from the bench to the bedside to effectively employ the misfolded proteins as promising therapeutic targets for cardiac diseases, but with trivial results. DESIGN: In this review, we describe the recent progresses in preclinical and clinical studies of protein misfolding and compromised protein quality control by selecting and reporting studies focusing on cardiovascular diseases including cardiomyopathies, cardiac amyloidosis, atherosclerosis, atrial fibrillation and thrombosis. RESULTS: In preclinical models, modulators of several molecular targets (eg heat shock proteins, unfolded protein response, ubiquitin protein system, autophagy and histone deacetylases) have been tested in various conditions with promising results although lacking an adequate transition towards clinical setting. CONCLUSIONS: At present, no therapeutic strategies have been reported to attenuate proteotoxicity in patients with CVD due to a lack of specific biomarkers for pinpointing upstream events in protein folding defects at a subclinical stage of the diseases requiring an intensive collaboration between basic scientists and clinicians.

Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation.

Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury.

Heart and Brain: Complex Relationships for Left Ventricular Dysfunction.

PURPOSE OF REVIEW: This review summarizes the evidence for the established vascular/hypoperfusion model and explores the new hypothesis that configures the heart/brain axis as an organ system where similar pathogenic mechanisms exploit physiological and pathological changes. RECENT FINDINGS: Although associated by common risk factors, similar epidemiological stratification and common triggers (including inflammation, oxidative stress, and hypoxia), heart failure and Alzheimer's disease have been, for long time, viewed as pathogenically separate illnesses. The silos began to be broken down with the awareness that vascular dysfunction, and loss of cardiac perfusion pump power, trigger biochemical changes, contributing to the typical hallmark of Alzheimer's disease (AD)-the accumulation of Aß plaques and hyperphosphorylated Tau tangles. Compromised blood flow to the brain becomes the paradigm for the "heart-to-head" connection. Compelling evidence of common genetic variants, biochemical characteristics, and the accumulation of Aß outside the brain suggests a common pathogenesis for heart failure (HF) and AD. These new findings represent just the beginning of the understanding the complex connection between AD and HF requiring further studies and interdisciplinary approaches. Altogether, the current evidence briefly summarized in this review, highlight a closer and complex relationship between heart failure and Alzheimer's that goes beyond the vascular/perfusion hypothesis. Genetic and biochemical evidence begin to suggest common pathogenic mechanisms between the two diseases involving a systemic defect in the folding of protein or a seeding at distance of the misfolded proteins from one organ to the other.

The Heart of the Alzheimer's: A Mindful View of Heart Disease.

Purpose of Review: This review summarizes the current evidence for the involvement of proteotoxicity and protein quality control systems defects in diseases of the central nervous and cardiovascular systems. Specifically, it presents the commonalities between the pathophysiology of protein misfolding diseases in the heart and the brain. Recent Findings: The involvement of protein homeostasis dysfunction has been for long time investigated and accepted as one of the leading pathophysiological causes of neurodegenerative diseases. In cardiovascular diseases instead the mechanistic focus had been on the primary role of Ca2+ dishomeostasis, myofilament dysfunction as well as extracellular fibrosis, whereas no attention was given to misfolding of proteins as a pathogenetic mechanism. Instead, in the recent years, several contributions have shown protein aggregates in failing hearts similar to the ones found in the brain and increasing evidence have highlighted the crucial importance that proteotoxicity exerts via pre-amyloidogenic species in cardiovascular diseases as well as the prominent role of the cellular response to misfolded protein accumulation. As a result, proteotoxicity, unfolding protein response (UPR), and ubiquitin-proteasome system (UPS) have recently been investigated as potential key pathogenic pathways and therapeutic targets for heart disease. Summary: Overall, the current knowledge summarized in this review describes how the misfolding process in the brain parallels in the heart. Understanding the folding and unfolding mechanisms involved early through studies in the heart will provide new knowledge for neurodegenerative proteinopathies and may prepare the stage for targeted and personalized interventions.

Primary cilia defects causing mitral valve prolapse.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.

Getting to the Heart of Alzheimer Disease.

In a somewhat narrow diagnostic lens, Alzheimer disease (AD) has been considered a brain-specific disease characterized by the presence of Aß (ß-amyloid) plaques and tau neural fibrillary tangles and neural inflammation; these pathologies lead to neuronal death and consequently clinical symptoms, such as memory loss, confusion, and impaired cognitive function. However, for decades, researchers have noticed a link between various cardiovascular abnormalities and AD-such as heart failure, coronary artery disease, atrial fibrillation, and vasculopathy. A considerable volume of work has pointed at this head to heart connection, focusing mainly on associations between cerebral hypoperfusion and neuronal degradation. However, new evidence of a possible systemic or metastatic profile to AD calls for further analysis of this connection. Aß aggregations-biochemically and structurally akin to those found in the typical AD pathology-are now known to be present in the hearts of individuals with idiopathic dilated cardiomyopathy, as well as the hearts of patients with AD. These findings suggest a potential systemic profile of proteinopathies and a new hypothesis for the link between peripheral and central symptoms of heart failure and AD. Herein, we provide an overview of the cardiovascular links to Alzheimer disease.

Gene Transfer to Rodent Hearts In Vivo.

Altering expression of specific genes in rodent models allows the dissection of various pathways involved in various disease states. Advances in gene transfer technologies produced vectors with tissue specificity, various time courses, increasing or knocking down genes in an externally controlled fashion. To achieve this goal, appropriate vector delivery method is essential for successfully conducting experiments. In this chapter, we focus on cardiac gene transfer in rodents that can be employed for delivering both viral and nonviral vectors.

Insights from Second-Line Treatments for Idiopathic Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is an independent nosographic entity characterized by left ventricular dilatation and contractile dysfunction leading to heart failure (HF). The idiopathic form of DCM (iDCM) occurs in the absence of coronaropathy or other known causes of DCM. Despite being different from other forms of HF for demographic, clinical, and prognostic features, its current pharmacological treatment does not significantly diverge. METHODS: In this study we performed a Pubmed library search for placebo-controlled clinical investigations and a post-hoc analysis recruiting iDCM from 1985 to 2016. We searched for second-line pharmacologic treatments to reconsider drugs for iDCM management and pinpoint pathological mechanisms. RESULTS: We found 33 clinical studies recruiting a total of 3392 patients of various durations and sizes, as well as studies that tested different drug classes (statins, pentoxifylline, inotropes). A metanalysis was unfeasible, although a statistical significance for changes upon treatment for molecular results, morphofunctional parameters, and clinical endpoints was reported. Statins appeared to be beneficial in light of their pleiotropic effects; inotropes might be tolerated more for longer times in iDCM compared to ischemic patients. General anti-inflammatory therapies do not significantly improve outcomes. Metabolic and growth modulation remain appealing fields to be investigated. CONCLUSIONS: The evaluation of drug effectiveness based on direct clinical benefit is an inductive method providing evidence-based insights. This backward approach sheds light on putative and underestimated pathologic mechanisms and thus therapeutic targets for iDCM management.

Aß Amyloid Pathology Affects the Hearts of Patients With Alzheimer's Disease: Mind the Heart.

BACKGROUND: Individually, heart failure (HF) and Alzheimer's disease (AD) are severe threats to population health, and their potential coexistence is an alarming prospect. In addition to sharing analogous epidemiological and genetic profiles, biochemical characteristics, and common triggers, the authors recently recognized common molecular and pathological features between the 2 conditions. Whereas cognitive impairment has been linked to HF through perfusion defects, angiopathy, and inflammation, whether patients with AD present with myocardial dysfunction, and if the 2 conditions bear a common pathogenesis as neglected siblings are unknown. OBJECTIVES: Here, the authors investigated whether amyloid beta (Aß) protein aggregates are present in the hearts of patients with a primary diagnosis of AD, affecting myocardial function. METHODS: The authors examined myocardial function in a retrospective cross-sectional study from a cohort of AD patients and age-matched controls. Imaging and proteomics approaches were used to identify and quantify Aß deposits in AD heart and brain specimens compared with controls. Cell shortening and calcium transients were measured on isolated adult cardiomyocytes. RESULTS: Echocardiographic measurements of myocardial function suggest that patients with AD present with an anticipated diastolic dysfunction. As in the brain, Aß40 and Aß42 are present in the heart, and their expression is increased in AD. CONCLUSIONS: Here, the authors provide the first report of the presence of compromised myocardial function and intramyocardial deposits of Aß in AD patients. The findings depict a novel biological framework in which AD may be viewed either as a systemic disease or as a metastatic disorder leading to heart, and possibly multiorgan failure. AD and HF are both debilitating and life-threatening conditions, affecting enormous patient populations. Our findings underline a previously dismissed problem of a magnitude that will require new diagnostic approaches and treatments for brain and heart disease, and their combination.

Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity.

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

Cofilin-2 phosphorylation and sequestration in myocardial aggregates: novel pathogenetic mechanisms for idiopathic dilated cardiomyopathy.

BACKGROUND: Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM), traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. This suggests a potential underlying cause for some of the iDCM cases. [Corrected] OBJECTIVES: This study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer's disease. METHODS: Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. RESULTS: Aggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease's morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of "stress-like" fibers and severely impaired cardiomyocyte contractility. CONCLUSIONS: Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies.

Protein post-translational modifications and misfolding: new concepts in heart failure.

A new concept in the field of heart-failure (HF) research points to a role of misfolded proteins, forming preamyloid oligomers (PAOs), in cardiac toxicity. This is largely based on few studies reporting the presence of PAOs, similar to those observed in neurodegenerative diseases, in experimental and human HF. As the majority of proteinopathies are sporadic in nature, protein post-translational modifications (PTMs) likely play a major role in this growing class of diseases. In fact, PTMs are known regulators of protein folding and of the formation of amyloid species in well-established proteinopathies. Proteomics has been instrumental in identifying both chemical and enzymatic PTMs, with a potential impact on protein mis-/folding. Here we provide the basics on how proteins fold along with a few examples of PTMs known to modulate protein misfolding and aggregation, with particular focus on the heart. Due to its innovative content and the growing awareness of the toxicity of misfolded proteins, an "Alzheimer's theory of HF" is timely. Moreover, the continuous innovations in proteomic technologies will help pinpoint PTMs that could contribute to the process. This nuptial between biology and technology could greatly assist in identifying biomarkers with increased specificity as well as more effective therapies.

Gene expression and genetic variation in human atria.

BACKGROUND: The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood. OBJECTIVE: The purpose of this study was to characterize gene expression and genetic variation in human atria. METHODS: We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0. RESULTS: We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a single nucleotide polymorphism at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. CONCLUSION: We found a distinct transcriptional profile between the right and left atrium and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Isolation, culture, and functional characterization of adult mouse cardiomyoctyes.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca(2+) dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.