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Biochemical Insights of Dr. Jose Eduardo de Salles Roselino

Posted in Biochemical pathways, Ca2+ triggered activation, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Curation, Developmental biology, Dialectic, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation and Evolution, Hematopoiesis, Historical relevance, Human Immune System in Health and in Disease, Interviews with Scientific Leaders, Lipid metabolism, Metabolism, Metabolomics, Na-K transport, Nephrology, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Nitric Oxide in Health and Disease, Synaptic vesicle, tagged cell surface interactions, cell-cell interactions, Cytoskeleton, cytoskeleton transport, epigenetic pathways, intercellular bridging, intracellular flux, intracellular localization, ion channels, Metabolism, protein conformation, protein modifications, protein-protein interaction networks, Proteins, quaternary structure, structural constraints, synaptic function on December 17, 2014| Leave a Comment »

Biochemical Insights of Dr. Jose Eduardo de Salles Roselino

Larry H. Bernstein, MD, FCAP, Interviewer, Curator

Leaders in Pharmaceutical Intelligence

Biochemical Insights of Dr. Jose Eduardo de Salles Roselino

http://pharmaceuticalintelligence.com/12/24/2014/larryhbern/Biochemical_
Insights_of_Dr._Jose_Eduardo_de_Salles_Roselino/

Article ID #165: Biochemical Insights of Dr. Jose Eduardo de Salles Roselino. Published on 12/17/2014

WordCloud Image Produced by Adam Tubman

Biochemical Insights of Dr. Jose Eduardo de Salles Roselino

How is it that developments late in the 20th century diverted the attention of
biological processes from a dynamic construct involving interacting chemical
reactions under rapidly changing external conditions effecting tissues and cell
function to a rigid construct that is determined unilaterally by the genome
construct, diverting attention from mechanisms essential for seeing the complete
cellular construct?

Larry, I assume that in case you read the article titled Neo – Darwinism, The
Modern Synthesis and Selfish Genes that bares no relationship with Physiology
with Molecular Biology J. Physiol 2011; 589(5): 1007-11 by Denis Noble, you might
find that it was the key factor required in order to understand the dislodgment
of physiology as a foundation of medical reasoning. In the near unilateral emphasis
of genomic activity as a determinant of cellular activity all of the required general
support for the understanding of my reasoning. The DNA to protein link goes
from triplet sequence to amino acid sequence. That is the realm of genetics.
Further, protein conformation, activity and function requires that environmental
and micro-environmental factors should be considered (Biochemistry). If that
were not the case, we have no way to bridge the gap between the genetic
code and the evolution of cells, tissues, organs, and organisms.

Consider this example of hormonal function. I would like to stress in
the cAMP dependent hormonal response, the transfer of information
that occurs through conformation changes after protein interactions.
This mechanism therefore, requires that proteins must not have their
conformation determined by sequence alone.
Regulatory protein conformation is determined by its sequence plus
the interaction it has in its micro-environment. For instance, if your
scheme takes into account what happens inside the membrane and
that occurs before cAMP, then production is increased by hormone
action. A dynamic scheme will show an effect initially, over hormone
receptor (hormone binding causing change in its conformation) followed
by GTPase change in conformation caused by receptor interaction and
finally, Adenylate cyclase change in conformation and in activity after
GTPase protein binding in a complex system that is dependent on self-
assembly and also, on changes in their conformation in response to
hormonal signals (see R. A Kahn and A. G Gilman 1984 J. Biol. Chem.
v. 259,n 10 pp6235-6240. In this case, trimeric or dimeric G does not
matter). Furthermore, after the step of cAMP increased production we
also can see changes in protein conformation. The effect of increased
cAMP levels over (inhibitor protein and protein kinase protein complex)
also is an effect upon protein conformation. Increased cAMP levels led
to the separation of inhibitor protein (R ) from cAMP dependent protein
kinase (C ) causing removal of the inhibitor R and the increase in C activity.
R stands for regulatory subunit and C for catalytic subunit of the protein
complex.
This cAMP effect over the quaternary structure of the enzyme complex
(C protein kinase + R the inhibitor) may be better understood as an
environmental information producing an effect in opposition to
what may be considered as a tendency towards a conformation
“determined” by the genetic code. This “ideal” conformation
“determined” by the genome would be only seen in crystalline
protein. In carbohydrate metabolism in the liver the hormonal signal
causes a biochemical regulatory response that preserves homeostatic
levels of glucose (one function) and in the muscle, it is a biochemical
regulatory response that preserves intracellular levels of ATP (another
function).
Therefore, sequence alone does not explain conformation, activity
and function of regulatory proteins.  If this important regulatory
mechanism was  not ignored, the work of  S. Prusiner (Prion diseases
and the BSE crisis Stanley B. Prusiner 1997 Science; 278: 245 – 251,
10 October) would be easily understood.  We would be accustomed
to reason about changes in protein conformation caused by protein
interaction with other proteins, lipids, small molecules and even ions.
In case this wrong biochemical reasoning is used in microorganisms.
Still it is wrong but, it will cause a minor error most of the time, since
we may reduce almost all activity of microorganism´s proteins to a
single function – The production of another microorganism. However,
even microorganisms respond differently to their micro-environment
despite a single genome (See M. Rouxii dimorphic fungus works,
later). The reason for the reasoning error is, proteins are proteins
and DNA are DNA quite different in chemical terms. Proteins must
change their conformation to allow for fast regulatory responses and
DNA must preserve its sequence to allow for genetic inheritance.

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Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Posted in Accountable Care Organizations, Affordable Care Act, Atherogenic Processes & Pathology, Best evidence, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Congenital Heart Disease, Curation, Curation methodology, Cytoskeleton, De novo synthesis, Dialectic, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Evolutionary cognition, Experimental validation, Explanatory, Federal Budget Appropriations, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Health Economics and Outcomes Research, Healthcare costs and reimbursement, HealthCare IT, Healthcare Reform, Historical relevance, HTN, Indigent Nutrition, Ionic Transporters Na+, K, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Medicare and Medicaid, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, PKC, Population Health Management, Genetics & Pharmaceutical, Pre-Clinical Animal Model Development, Regenerative Biology and Medicine, Skilled Nursing Facilities, Stem Cells for Regenerative Medicine, Synaptic vesicle, Technology Advance Assessment of, Technology Capital Expenses, Technology Transfer: Biotech and Pharmaceutical, Theoretic convergence, Translational Effectiveness, Translational Research, Translational Science, Uninsured and Underinsured, tagged 249 SNPs associated with a number of heart-related conditions and treatments., Accountable Care Organization, acute coronary syndromes, Acute decompensated heart failure, Affordable Care Act, atherogenesis, Atherosclerosis, Atrial Fibrillation: IL6R Polymorphism in Whites and African Americans, calcium calmodulin kinase, calcium efflux, Cardiac & Vascular Repair, cardiac arrhythmias, cardiac biomarkers, Cardiogenesis, cardiomyocyte, cardiomyocyte hypertrophy, Content Curation, Coronary artery disease, Coronary circulation, Curation, efflux transporter, financial cost accounting, ion channels, ion transport, Na-K-Cl cotransporter, PKC, Pyruvate Kinase, seven CYP2C19 SNPs linked to Plavix response, SNPs, Synaptic vesicle on January 2, 2014| Leave a Comment »

Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN

and

Content Consultant to Six-Volume e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

This portion summarises what we have covered and is now familiar to the reader. There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading. This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology. It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process. In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views. There were surprises, and there were unfulfilled answers along the way. The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable. This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified. In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.

We introduced the content as follows:

1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.

Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.

Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
PCI for valves vs Open heart Valve replacement
PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.

2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.

3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.

4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.

Curation

Curation is an active filtering of the web’s and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view. Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale. One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive. The same source says “Scoop.it is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.

Healthcare and Affordable Care Act

We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate. But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system. There has been a very large rush and an extension to sign up. There are many features that we can take note of:

1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.

2. These changes that are notable are:

The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
This is not helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.

3. The medical care system is affected by the issues above, but the complexity is not to be discounted.

The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
Medicaid is expanded substantially under the new ACA.
The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.

Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.

This portion is outlined as follows:

2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease	2.2 The Role of Calcium in Health and Disease	2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease
Genomics & Genetics of Cardiovascular Disease Diagnoses	actin cytoskeleton	wall stress, ventricular workload, contractile reserve
Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging	calcium and actin skeleton, signaling, cell motility	hypertension & vascular compliance
Genetics of Conduction Disease	Ca+ stimulated exostosis: calmodulin & PKC (neurotransmitter)	complications & MVR
	disruption of Ca2+ homeostasis cardiac & vascular smooth muscle
	synaptotagmin as Ca2+ sensor & vesicles
	atherosclerosis & ion channels

It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system. The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence. This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”. The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression. This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically. The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.

Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm. The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction. The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction. That is only part of the story. Ion transport occurs into or out of the cell, the latter termed exostosis. Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles. This also involves K+-Na+-ATPase. The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells. When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial. The glass is half full!

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Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Posted in Cardiovascular Pharmacogenomics, Diabetes Mellitus, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Ionic Transporters Na+, K, Molecular Genetics & Pharmaceutical, Na-K transport, Na-K-ATPase, Nitric Oxide in Health and Disease, Origins of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Proteomics, tagged ATO2A@, ATPase, Aviva Lev-Ari, CAD, Coronary artery disease, Coronary circulation, coronary microvascular dysfunction, eNOS, genetic polymorphisms, Heart disease, Heart Failure, Hebrew University of Jerusalem, ion channels, ischemic heart diases (IHD), KCN5A gene (Kv1.5 channel), Kir6, Myocardial Ischemia, Nav 1.5, non-modifiable protective factor, NOS encoding, rs5215_GG, rs5219_AA, SCN5A, Thomas C. Südhof, voltage-gated K ion channel on December 21, 2013| Leave a Comment »

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Reviewer and Co-Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Image created by Adina Hazan 06/30/2021

The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studied by our Team in 2012 and 2013. This is article TWELVE in a 13 article series listed at the end of this article.

Chiefly, our sources of inspiration were the following:

1. 2013 Nobel work on vesicles and calcium flux at the neuromuscular junction

Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells

The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells. This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell. Specificity in the delivery of molecular cargo is essential for cell function and survival.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/advanced-medicineprize2013.pdf

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

2. Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

http://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

3. Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine

Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells. His work led to the discovery that specific steroids that have crucial roles, as the regulation of cell viability, heart contractility, blood pressure and brain function. His research has implications for the fundamental understanding of body functions, as well as for several pathological states such as heart failure, hypertension and neurological and psychiatric diseases.

Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/18/physiologist-professor-lichtstein-chair-in-heart-studies-at-the-hebrew-university-elected-dean-of-the-faculty-of-medicine-at-the-hebrew-university-of-jerusalem/

4. Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

5. Seminal Curations by Dr. Aviva Lev-Ari on Genetics and Genomics of Cardiovascular Diseases with a focus on Conduction and Cardiac Contractility

Atrioventricular (AV) Conduction Disease (block): Human Mutations affecting the Voltage Clock

Aviva Lev-Ari, PhD, RN

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Aviva Lev-Ari, PhD, RN

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013

Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Other related research by the Team of Leaders in Pharmaceutical Business Intelligence published on the Open Access Online Scientific Journal

http://pharmaceuticalintelligence.com

See References to articles at the end of this article on

ION CHANNEL and Cardiovascular Diseases

http://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases – The Role of Calcium Calmodulin Kinase (CKCaII) and Ca(2) flux

Mitochondria and Oxidative Stress Role in Cardiovascular Diseases

Thus, the following article follows a series of articles on ion-channels and cardiac contractility mentioned, above. The following article is closely related to the work of Prof. Lichtstein.

These investigators studied the possible correlation between

Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and
single-nucleotide polymorphisms (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including
ion channels acting in vascular smooth muscle and/or
endothelial cells of coronary arteries.

They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as

the whole coding region of KCN5A gene (Kv1.5 channel).

The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be used

to identify individuals at relatively low-risk for cardiovascular disease
an independent protective role of the
- rs5215_GG against developing CAD and
- a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction

Their findings are a lead into further investigations on ion channels and IHD affecting the microvasculature.

Role of genetic polymorphisms of ion channels in the pathophysiology of coronary microvascular dysfunction and ischemic heart disease

BasicResCardiol(2013)108:387 http//dx.dio.org/10.1007/s00395-013-0387-4

F Fedele1•M Mancone1•WM Chilian2•P Severino2•E Canali•S Logan•ML DeMarchis3•M Volterrani4•R Palmirotta3•F Guadagni3

1Department of Cardiovascular, Respiratory, Nephrology, Anesthesiology and Geriatric Sciences,Sapienza University of Rome, UmbertoI Policlinic, Rome, Italy e-mail:francesco.fedele@uniroma1.it
2Department of Integrative Medical Sciences, Northeastern Ohio Universities College of Medicine, Rootstown,OH
3Department of Advanced Biotechnologies and Bioimaging, IRCCS San Raffaele Pisana,Rome,It
4Cardiovascular Research Unit, Department of Medical Sciences, Centre for Clinical and Basic Research, Raffaele Pisana, Rome, Italy (CBFR)

BasicResCardiol(2013)108:387 http//dx.dio.org/10.1007/s00395-013-0387-4
This article is published with open access at Springerlink.com

Abstract

Conventionally,ischemic heart disease (IHD) study is equated with large vessel coronary disease (CAD). However, recent evidence has suggested

a role of compromised microvascular regulation in the etiology of IHD.

Because regulation of coronary blood ﬂow likely involves

activity of speciﬁc ion-channels, and
key factors involved in endothelium-dependent dilation,

genetic anomalies of ion-channels or speciﬁc endothelial-regulators may underlie coronary microvascular disease.

We aimed to evaluate the clinical impact of single-nucleotide polymorphisms in genes encoding for

ion-channels expressed in the coronary vasculature and the possible
correlation with IHD resulting from microvascular dysfunction.

242 consecutive patients who were candidates for coronary angiography were enrolled. A prospective, observational, single-center study was conducted,

analyzing genetic polymorphisms relative to

(1) NOS3 encoding for endothelial nitric oxide synthase (eNOS);
(2) ATP2A2 encoding for the Ca/H-ATP-ase pump (SERCA);
(3) SCN5A encoding for the voltage-dependent Na channel (Nav1.5);
(4) KCNJ8 and in KCNJ11 encoding for the Kir6.1and Kir6.2 subunits
of genetic K-ATP channels, respectively;and
(5) KCN5A encoding for the voltage-gated K channel (Kv1.5).

No signiﬁcant associations between clinical IHD manifestations and

polymorphisms for SERCA, Kir6.1, and Kv1.5. were observed (p[0.05),

whereas speciﬁc polymorphisms detected in eNOS, as well as in Kir6.2 and Nav1.5 were found to be correlated with

IHD and microvascular dysfunction.

Interestingly, genetic polymorphisms of ion-channels seem to have an important clinical impact

inﬂuencing the susceptibility for microvascular dysfunction and (IHD,
independent of the presence of classic cardiovascular risk factors: atherosclerosis

http//dx.dio.org/10.1007/s00395-013-0387-4

Keywords: Ion-channels, Genetic polymorphisms, Coronary microcirculation, Endothelium, Atherosclerosis Ischemic heart disease

Introduction

Historically, in the interrogation of altered vascular function in patientswith ischemic heart disease (IHD), scientists have focused their attention on the correlation between

endothelial dysfunction and
atherosclerosis [11, 53, 65, 67].

The endothelium-independent dysfunction in coronary microcirculation and its possible correlations with

atherosclerotic disease and
myocardial ischemia has not been extensively investigated.

In normal conditions, coronary blood ﬂow regulation (CBFR) is mediated by several different systems, including

endothelial,
nervous,
neurohumoral,
myogenic, and
metabolic mechanisms [2, 10, 14, 15, 63, 64, 69].

Physiologic CBFR depends also on several ion channels, such as

ATP-sensitive potassium (KATP) channels,
voltage-gated potassium (Kv) channels,
voltage-gated sodium (Nav) channels, and others.

Ion channels regulate the concentration of calcium in both

coronary smooth muscle and endothelial cells, which
modulates the degree of contractile tone in vascular muscle and
the amount of nitric oxide that is produced by the endothelium

Ion channels play a primary role in the rapid response of both

the endothelium and vascular smooth muscle cells of coronary arterioles
to the perpetually ﬂuctuating demands of the myocardium for blood ﬂow
[5, 6, 13, 18, 33, 45, 46, 51, 52, 61, 73, 75].

Despite this knowledge, there still exists an important gap about

the clinical relevance and
causes of microvascular dysfunction in IHD.

By altering the overall

regulation of blood ﬂow in the coronary system,
microvascular dysfunction could alter the normal distribution of shear forces in large coronary arteries

Proximal coronary artery stenosis could

contribute to microvascular dysfunction [29, 60]. But
ion channels play a critical role in microvascular endothelial
and smooth muscle function.

Therefore, we hypothesized that alterations of coronary ion channels could be the primary cause in a chain of events leading to

microvascular dysfunction and
myocardial ischemia,

independent of the presence of atherosclerosis.

Therefore, the objective of our study was to evaluate the possible correlation between

IHD and single-nucleotide polymorphisms (SNPs) for genes encoding several regulators involved in CBFR, including
ion channels acting in vascular smooth muscle and/or
endothelial cells of coronary arteries.

Discussion

Implications of the present work. This study describes the possible correlation of polymorphisms in genes encoding for CBFR effectors (i.e., ion channels, nitric oxide synthase, and SERCA) with the susceptibility for microcirculation dysfunction and IHD.

Our main findings are as follows: (Group 3 – Normal Patients – anatomically and functionally normal coronary arteries).

In Group 3, the genotype distribution of SNP rs5215 (Kir6.2/KCNJ11) moderately deviates from the HW equilibrium (p = 0.05).
In Group 1 (CAD), the polymorphism rs6599230 of Nav1.5/SCN5A showed deviation from HW equilibrium (p = 0.017).
The genotypic distribution of rs1799983 polymorphism for eNOS/NOS3 is inconsistent with the HW equilibrium in groups 1, 2, and 3 (p = 0.0001, p = 0.0012 and p = 0.0001, respectively).

Haplotype analyses revealed that there is no linkage disequilibrium between polymorphisms of the analyzed genes. There was no significant difference in the prevalence of T2DM (p = 0.185) or dyslipidemia (p = 0.271) between groups, as shown in Table2. In regards to genetic characteristics, no significant differences between the three.

1. A marked HW disequilibrium in the genotypic distribution of rs1799983 polymorphism for eNOS/NOS3 was observed in all three populations. Moreover, this SNP seems to be an independent risk factor for microvascular dysfunction, as evidenced by multivariate analysis;
2. The SNPs rs5215_GG, rs5218_CT, and rs5219_AA for Kir6.2/KCJ11 could reduce susceptibility to IHD, since they were present more frequently in patients with anatomically and functionally normal coronary arteries;
3. In particular, with regard to rs5215 for Kir6.2/KCJ11, we observed a moderate deviation from the HW equilibrium in the genotypic
distribution in the control group. In addition, this genotype appears to be an independent protective factor in the development of IHD, as evidenced by multivariate analysis;
4. Furthermore, the trend observed for the SNP rs5219_AA of Kir6.2/KCNJ11 may suggest an independent protective factor in the development of coronary microvascular dysfunction
5. The rs1805124_GG genotype of Nav1.5/SCN5A seems to play a role against CAD;
6. No association seems to exist between the polymorphisms of SERCA/ATP2A2, Kir6.1/KCNJ8, and Kv1.5/KCNA5 and the presence of IHD;
7. All groups are comparable regarding the cardiovascular risk factors of T2DM and dyslipidemia, illustrating a potentially important implication of genetic polymorphisms in the susceptibility to IHD.

It is important to underline that the control group (Group 3) is a high-risk population, because of their cardiovascular risk factors

hypertension = 17 %,
T2DM = 34.1 %,
dyslipidemia = 41.4 %,

with an appropriate indication for coronary angiography, in accordance with current guidelines. Nevertheless, these patients were demonstrated to have both anatomically and functionally normal coronary arteries. Moreover, as shown in Tables 2 and 3, we observed that

rs5215_GG, rs5218_CT and rs5219_AA for Kir6.2/KCNJ11 had a higher prevalence in this group,compared to patients with CAD
and patients with microvascular dysfunction.

Moreover, as shown in Table 4, the presence of the rs5215_GG polymorphism for the Kir6.2 subunit was

inversely correlated with the prevalence of cardiovascular risk factors and CAD,whereas
rs5219_AA of the Kir6.2 subunit trended towards an inverse correlation with coronary microvascular dysfunction.

On the other hand, the SNP rs1799983_GT of eNOS was

confirmed to be an independent risk factor for microvascular dysfunction.

Our data suggest that the presence of certain genetic polymorphisms may represent a non-modifiable protective factor that could be used

to identify individuals at relatively low-risk for cardiovascular disease,
regardless of the presence of T2DM and dyslipidemia.

Current Clinical and Research Context

In normal coronary arteries, particularly the coronary microcirculation, there are several different mechanisms of CBFR, including

endothelial, neural, myogenic, and metabolic mediators [2, 8, 10, 12, 14, 15, 37, 55, 63, 64, 69].

In particular, endothelium-dependent vasodilation acts mainly via eNOS-derived nitric oxide (NO) in response to acetylcholine and shear stress.

NO increases intracellular cyclic guanosine monophosphate. It also causes vasodilation via

activation of both K-Ca channels and K-ATP channels.

Recent data suggested a pathophysiologically relevant role for the polymorphisms of eNOS/NOS3 in human coronary vasomotion [40–43]. Our data suggest that rs1799983_GT at exon 7 (Glu298Asp, GAG-GAT) of eNOS/NOS3 represents

an independent risk factor for coronary micro-vascular dysfunction, which agrees with a recent meta-analysis reporting an
association of this SNP with CAD in Asian populations [74]. In addition,
this SNP has been associated with endothelial dysfunction, although the mechanisms are not well defined [30].

Consistently, a recent study performed on 60 Indian patients with documented history of CAD reported a significantly higher frequency of rs1799983 (p.05) compared to control subjects, indicating that

variations in NOS3 gene may be useful clinical markers of endothelial dysfunction in CAD [54].

Interestingly, another association between rs1799983_GT and impaired collateral development has been observed in patientswith a

high-grade coronary stenosis or occlusion [19].

As is well known, the significance of the mechanisms of CBFR is partly determined by the location within the coronary vasculature. For instance, for vessels with a diameter of < 200 µm—which comprise the coronary microcirculation—metabolic regulation of coronary blood flow is considered the most important mechanism [24, 63]. Importantly, many of these mediators of metabolic regulation act through specific ion channels. In particular, in both coronary artery smooth muscle cells and endothelial cells

potassium channels determine the resting membrane potential (Em) and serve as targets of endogenous and therapeutic vasodilators [9, 27].

Several types of K+ channels are expressed in the coronary tree.

The K-ATP channels couple cell metabolic demand to conductance, via pore-forming (Kir6.1 and/or Kir6.2) subunits and regulatory
[sulphonylurea-binding (SUR 1, 2A, or 2B)] subunits.
Kir6.x allows for channel inhibition by ATP, while SURx is responsible for channel activation by ADP and Mg2+.

K-ATP channel activation results in an outward flux of potassium and

consequent hyperpolarization, resulting in
voltage-gated calcium channel closure,
decreased Ca2+ influx, and ultimately
vasodilation [1, 5, 18, 20, 21, 33, 61, 62, 73, 75].

Our data do not support any significant difference regarding the Kir6.1 subunit of the K-ATP channel. On the other hand, this study suggests

an important role of specific SNPs for the Kir6.2 subunit (Tables 2, 3)—i.e., rs5215, rs5219, and rs5218—

in the susceptibility to IHD and microvascular dysfunction. These SNPs are among the most studied K-ATP channel polymorphisms, especially in the context of diabetes mellitus. In fact, in both Caucasian and Asian populations, these three SNPs as well as other genetic polymorphisms for the KCNJ11 gene have been associated with diabetes mellitus [34, 35, 44, 50, 57, 58, 70].

Nevertheless, the precise

structure–function impacts of the various amino acid substitutions remain unclear.

The rs5215 and rs5219 polymorphisms, also known as I337V and E23K, respectively, are highly linked with reported

concordance rates between 72 and 100 % [22, 23, 56].

The high concordance between rs5219 and rs5215 suggests that these polymorphisms

may have originated in a common ancestor, further indicating a
possible evolutionary advantage to their maintenance in the general population [49].

In our study, multivariate analysis suggests both an independent protective role of the

rs5215_GG against developing CAD and
a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction (Table 4a, b).
The variant rs5215_GG is a missense SNP located in the gene KCNJ11 at exon 1009 (ATC-GTC) and results in
the substitution of isoleucine (I) residue with valine (V) [23].

Future studies are necessary to better understand the influence of this single amino acid variant on the function of the channel.

In humans, vasodilation of the coronary microvasculature in response to hypoxia and K-ATP channel opening

are both impaired in diabetes mellitus [39].

It is also described that gain-of-function mutations of the KCNJ11 gene cause neonatal diabetes mellitus, and loss-of-function mutations lead to congenital hyperinsulinism [43]. Our study is not discordant with previous studies about the correlation of SNPs of the Kir6.2 subunit and diabetes mellitus. Rather, our findings show that these SNPs are correlated with anatomically and functionally normal coronary arteries,

independent of the presence of either diabetes mellitus or dyslipidemia.

These data suggest the possibility that these particular SNPs may identify individuals with decreased risk for coronary microcirculatory dysfunction and IHD,

regardless of the presence of T2DM and/or dyslipidemia.

However, further studies are necessary to confirm these findings. In this context, to better investigate the implications of genetic variation in the K-ATP channel,

future studies should include ion channel’s functional modification due to the SNPs and analysis of SUR subunits.

More than 40-kV channel subunits have been identified in the heart, and sections of human coronary smooth muscle cells demonstrate Kv1.5 immunoreactivity [16, 17, 27, 38]. Through constant regulation of smooth muscle tone, Kv channels contribute to the control of coronary microvascular resistance [4, 7]. Pharmacologic molecules that inhibit Kv1.5 channels such as

pergolide [25],
4-amino-pyridine [32], and
correolide [17]

lead to coronary smooth muscle cell contraction and block the coupling between

cardiac metabolic demand and
coronary blood flow.

However, no significant differences were identified between the study groups in terms of the particular polymorphisms for Kv1.5 that were analyzed in this study. Expression of

the voltage-dependent Na+ channel (Nav) has been demonstrated in coronary microvascular endothelia cells [3, 66].

Our analysis reveals a possible implication of the polymorphism rs1805124_GG for Nav1.5 channel with the presence of anatomically and functionally normal coronary arteries. This SNP leads to a homozygous 1673A-G transition, resulting in a His558-to-Arg (H558R) substitution. It is important to underline that

our data are the first to correlate the polymorphism rs1805124_GG with IHD.

Further research is necessary to confirm the observed implication.

Finally, we have analyzed the sarco/endoplasmic reticulum calcium transporting Ca2+-ATPase (SERCA), which is fundamental in the regulation of intracellular Ca2+ concentration [6].

SERCA is an intracellular pump that

catalyzes the hydrolysis of ATP coupled with the
translocation of calcium from the cytosol into the lumen of the sarcoplasmic reticulum.

Although this pump plays a critical role in regulation of the contraction/relaxation cycle, our analysis did not reveal any apparent association between

genetic variants of SERCA and the
prevalence of microvascular dysfunction or IHD.

Conclusions

This pilot study is the first to compare the prevalence of SNPs in genes encoding coronary ion channels between patients

with CAD or microvascular dysfunction and those with both anatomically and functionally normal coronary arteries.

Taken together, these results suggest the possibility of associations between SNPs and IHD and microvascular dysfunction, although

the precise manners by which specific genetic polymorphisms affect ion channel function and expression

have to be clarified by further research involving larger cohorts.

Limitations and future perspectives

Notable limitations of this pilot study are as follows:

1. Due to the lack of pre-existing data, the power calculation was performed in advance on the basis of assumptions of allele frequencies and the population at risk.

2. The sample size for each group is small, mainly due to both the difficulty in enrolling patients with normal coronary arteries and normal microvascular function (group 3) and the elevated costs of the supplies such as Doppler flow wires.

3. There is a lack of ethnic diversity of our cohort.

4. Currently, there is an absence of supportive findings in another independent cohort or population. However, our pilot study included patients within a well-defined, specific population and was aimed to identify the presence of statistical associations between selected genetic polymorphisms and the prevalence of a specific disease.

5. There is a lack of functional characterization of the described genetic polymorphisms.

6. We have not identified any correlation between novel SNPs and IHD. Nevertheless, we completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as the whole coding region of KCN5A gene (Kv1.5 channel). Moreover, we examined previously described SNPs since there are no data in the literature regarding the possible association of the prevalences of those polymorphisms in the examined population.More extensive studies are necessary to confirm our findings, possibly with a larger number of patients. Future investigations are also required to confirm the roles of ion channels in the pathogenesis of coronary microvascular dysfunction and IHD. These studies should involve analysis of both other subunits of the K-ATP channels

sulfonylurea receptor, SURx and further coronary ion channels (e.g., calcium-dependent K channels), as well as
in vitro evaluation of ion channel activity by patch clamp and analysis of channel expression in the human cardiac tissue.

Moreover, to better address the significance of microvascular dysfunction in IHD, it could be interesting to analyze

typical atherosclerosis susceptibility genes (e.g., PPAP2B, ICAM1, et al.).

Methods

In this prospective, observational, single-center study – 242 consecutive patients admitted to our department were enrolled with

the indication to undergo coronary angiography .

All patients matched inclusion criteria

age [18];
suspected or documented diagnosis of acute coronary syndrome or stable angina
with indication(s) for coronary angiography, in accordance with current guidelines [36, 68], and
the same ethno-geographic Caucasian origin) and

Exclusion Criteria

previous allergic reaction to iodine contrast,
renal failure,
simultaneous genetic disease,
cardiogenic shock,
non- ischemic cardiomyopathy

All patients signed an informed consent document –

prior to participation in the study, which included

acknowledgement of the testing procedures to be performed
(i.e., coronary angiography; intracoronary tests; genetic analysis, and processing of personal data).

The study was approved by the Institution’s Ethics Committee.
All clinical and instrumental characteristics were collected in a dedicated database.

Study Design

(a) Standard therapies were administered, according to current guidelines [36, 68].
(b) An echocardiography was performed before and after coronary angiography
(c) Coronary angiography was performed using radial artery or femoral artery
Judkins approach via sheath insertion.
(d) In patients showing normal epicardial arteries, intracoronary functional tests
were performed through Doppler ﬂow wire to evaluate

both endothelium-dependent microvascular function
[via intracoronary (IC) infusion of acetylcholine (2.5–10 lg)] and
nonendothelium-dependent microvascular function
[via IC infusion of adenosine (5 lg)] [31].

(e) In all enrolled patients, a peripheral blood sample for genetic analysis was taken.

On the basis of the coronary angiography and the intracoronary functional tests,

the 242 patients were divided into three groups (see also Fig. 1).

Group 1: 155 patients with anatomic coronary alteration
(comprising patients with acute coronary syndrome and chronic stable angina).
- microvascular dysfunction deﬁned as coronary ﬂow reserve (CFR) \ 2.5
- after IC infusion of acetylcholine and adenosine].
Group 2: 46 patients with functional coronary alteration
[normal coronary arteries as assessed by angiography, and
- as assessed by angiography and with normal functional tests
  (CFR C 2.5 after intracoronary infusion of acetylcholine and adenosine) (Fig. 1).
Group 3: 41 patients with anatomically and functionally normal coronary arteries

Fig. 1 Study design: 242 consecutive not randomized patients matching inclusion and exclusion criteria were enrolled.
In all patients, coronary angiography was performed, according to current ESC/ACC/AHA guidelines. In patients with
angiographically normal coronary artery, intracoronary functional tests were performed. In 242 patients
(155 with coronary artery disease, 46 patients with micro-vascular dysfunction, endothelium and/or non-endothelium
dependent, and 41 patients with anatomically and functionally normal coronary arteries) genetic analysis was performed.

Genetic Analysis

In conformity with the study protocol, ethylenediaminetetraacetic acid (EDTA) whole blood samples were collected according
to the international guidelines reported in the literature [48]. Samples were transferred to the Interinstitutional Multidisciplinary
BioBank (BioBIM) of IRCCS San Raffaele Pisana (Rome) and stored at -80 C until DNA extraction. Bibliographic research by
PubMed and web tools OMIM (http://www.ncbi.nlm.nih.gov/omim), Entrez SNP (http://www.ncbi.nlm.nih.gov/snp), and
Ensembl (http://www.ensembl.org/index.html) were used to select variants of genes involved in signaling pathways

related to ion channels and/or reported to be associated with
microvascular dysfunction and/or myocardial ischemia and/or
diseases correlated to IHD, such as diabetes mellitus.

Polymorphisms for the following genes were analyzed:

NOS3 (endothelial nitric oxide synthase, eNOS),
ATP2A2 (Ca2+/H+-ATPase pump, SERCA2),
SCN5A (voltage-dependent Na+ channel,
Nav1.5),
KCNJ11 (ATP-sensitive K+ channel, Kir6.2 subunit),
KCNJ8 (ATP-sensitive K+ channel, Kir6.1 subunit) and
KCNA5 (voltage-gated K+ channel, Kv1.5).

In particular, we completely analyzed by direct sequencing

exon 3 of KCNJ8 (Kir6.1 subunit), which includes eight SNPs, as well as
the whole coding region of KCNA5 (Kv1.5 channel), which includes 32 SNPs and
four previously described variants [26, 47, 71, 72].

We also examined

the whole coding region of KCNJ11 (Kir6.2 subunit), for which sequence variants are described [26, 28].

All SNPs and sequence variants analyzed—a total of 62 variants of 6 genes—are listed in Table 1.

BRC 2013 fedele genetic polymorphisms of ion channels_page_004

BRC 2013 fedele genetic polymorphisms of ion channels_page_005

DNA was isolated from EDTA anticoagulated whole blood using the MagNA Pure LC instrument and theMagNA Pure LC
total DNA isolation kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Standard
PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems, CA) using HotStarTaq Master Mix
(HotStarTaq Master Mix Kit, QIAGEN Inc, CA). PCR conditions and primer sequences are listed in Table 1.

In order to exclude preanalytical and analytical errors, all direct sequencing analyses were carried out on both
strands using Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), run on an ABI 3130
Genetic Analyzer (Applied Biosystems), and repeated on PCR products obtained from new nucleic acid extractions.
All data analyses were performed in a blind fashion.

Statistical Analysis

This report, intended as pilot study, is the ﬁrst to compare

the prevalence of SNPs in genes encoding several effectors (including ion channels)
involved in CBFR between these groups of patients.

No deﬁnite sample size could be calculated to establish a power analysis. groups of patients. However, assuming

a 15 % prevalence of normal macrovascular and microvascular coronary ﬁndings in unselected patients
undergoing coronary angiography,

we estimated that

a sample size of at least 150 patients could enable the computation of two-sided 95 % conﬁdence intervals for
- such prevalence estimates ranging between -5.0 and + 5.0 %.

The signiﬁcance of the differences of observed alleles and genotypes between groups, as well as

analysis of multiple inheritance models for SNPs were also tested
(co-dominant, dominant, recessive, over-dominant and log-additive)
using a free web-based application (http://213. 151.99.166/index.php?module=Snpstats)
designed from a genetic epidemiology point of view to analyze association studies.

Akaike Information Criterion (AIC) was used to determine the best-ﬁtting inheritance model for analyzed SNPs,

with the model with the lowest AIC reﬂecting the best balance of goodness-of-fit and parsimony.

Moreover, the allelic frequencies were estimated by gene counting, and the genotypes were scored. For each gene,

the observed numbers of each genotype were compared with those expected for a population in Hardy–Weinberg (HW) equilibrium
using a free web-based application (http://213.151.99.166/index.php?module=Snpstats) [59].

Linkage disequilibrium coefﬁcient (D0) and haplotype analyses were assessed using the Haploview 4.1 program.
Statistical analysis was performed using SPSS software package for Windows v.16.0 (SPSS Inc., Chicago, IL).

All categorical variables are expressed as percentages, and all continuous variables as mean ± standard deviation.
Differences between categorical variables

were analyzed by Pearson’s Chi-SQ test.

Given the presence of three groups, differences between continuous variables, were calculated using
(including the number of SNPs tested),

one-way ANOVA; a post-hoc analysis with Bonferroni correction was made for multiple comparisons.

Univariate and multivariate logistic regression analyses

the independent impact of genetic polymorphisms on
- coronary artery disease and microvascular dysfunction,

were performed to assess the independent impact of

genetic polymorphisms on coronary artery disease
and microvascular dysfunction,

while adjusting for other confounding variables. The following parameters were entered into the model:

age,
male gender,
type 2 diabetes mellitus (T2DM),
systemic arterial hypertension,
dyslipidemia,
smoking status, and
family history of myocardial infarction (MI).

Only variables with a p value < 0.10 after univariate analysis were entered

into the multivariable model as covariates.

A two-tailed p < 0.05 was considered statistically signiﬁcant.

Deﬁnition of Cardiovascular Risk Factors

Patients were classiﬁed as having T2DM if they had

fasting levels of glucose of >126 mg/dL in two separate measurements or
if they were taking hypoglycemic drugs.

Systemic arterial hypertension was deﬁned as

systolic blood pressure > 140 mmHg / diastolic blood pressure > 90 mmHg
in two separate measurements or
if the patient was currently taking antihypertensive drugs.

Dyslipidemia was considered to be present if

serum cholesterol levels were>220 mg/dL or
if the patient was being treated with cholesterol-lowering drugs.

Family history of MI was defined as a first-degree relative with MI before the age of 60 years.

Results

Sixty-two polymorphisms distributed among six genes coding for

nitric oxide synthase,
the SERCA pump, and
ion channels
- were screened for sequence variations using PCR amplification and
- direct DNA sequencing analysis

in the population of

155 patients with CAD (group 1),
46 patients with microvascular dysfunction (group 2), and
41 patients with normal coronary arteries and
- normal endothelium dependent and endothelium-independent vasodilation (group 3).

In Group 3, the genotype distribution of

SNP rs5215 (Kir6.2/KCNJ11) moderately deviates from the HW equilibrium (p = 0.05).

In Group 1 (CAD), the polymorphism

rs6599230 of Nav1.5/SCN5A showed deviation from HW equilibrium (p = 0.017).

The genotypic distribution of groups in terms of polymorphisms for

eNOS/NOS3, SERCA/ATP2A2, Nav1.5/SCN5A, Kir6.1/KCNJ8, or Kv1.5/KCNA5

were noticed. However, significant differences (p.05) for the SNPs

rs5215_GG, and
rs5219_AA of Kir6.2/KCNJ11 were observed,

as shown in Table 2.

Table 3 displays significant differences between normal subjects (group 3) and

patients with either CAD (group 1) or microvascular dysfunction (group 2).

When correcting for other covariates as risk factors, the rs5215_GG genotype of Kir6.2/KCNJ11 was found to be

significantly associated with CAD after multivariate analysis (OR = 0.319, p = 0.047, 95 % CI = 0.100–0.991), evidencing
a ‘‘protective’’ role of this genotype, as shown in Table 4a.

Similarly, a trend that supports this role of Kir6.2/KCNJ11 was also observed

in microvascular dysfunction for rs5219 AA. In contrast,
rs1799983_GT for eNOS/NOS3 was identified as an independent risk factor

following multivariate analysis (Table 4b), which agrees with literature findings as described below.

SOURCE for TABLES

BasicResCardiol(2013)108:387 http//dx.dio.org/10.1007/s00395-013-0387-4

Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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Basic Res Cardiol (2013) 108:387 http://dx.doi.org/10.1007/s00395-013-0387-4

ION CHANNEL and Cardiovascular Diseases

http://pharmaceuticalintelligence.com/?s=Ion+Channel

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Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

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Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

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Human embryonic pluripotent stem cells and healing post-myocardial infarction

Posted in Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, Resident-cell-based, tagged biomarker, cardiomyocyte, Cardiovascular disease, Cell potency, heart pailure, ion channels, Larry H. Bernstein, MI, myocardial infarction, Nebivolol, New England Journal Medicine, pluripotent stem cells, reprogramming, transplantation on August 7, 2012| 7 Comments »

Human embryonic pluripotent stem cells and healing post-myocardial infarction

Curator: Larry H. Bernstein, MD, FCAP
I present a followup based on several recent posts related to the promise of using induced human pluripotent stem cells for repair of ischemia damaged myocardium postinfarct and related effect of heart failure (HF). There has been a change in the concept of cardiovascular risk related to the emergent knowledge of the biology underlying oxidative stress. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual’s propensity for future cardiovascular events (1). Vascular injury and repair are significantly mediated by circulating endothelial progenitor cells (1). Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR) which are measured by pre-enrichment flow cytometry with specific identification of cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) (2), used in the assessment of various diseases and physiological states. Improvements in flow cytometry include the Attune® cytometer, which enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes (3). Using these methods of analyses, it became evident that circulating endothelial progenitor cells have angiogenic potential.

Activators and inhibitors have been tested for their ability to modulate angiogenesis in early phase clinical trials, and in the case of anti-Flk1 antibodies clinical utility has been demonstrated for anti-tumor strategies (4). Extending this concept further, we pose that just as the progenitor role invoked for angiogenesis, transcriptional networks and interactions are involved in the morphogenesis of the developing vertebrate heart. The identities of crucial regulators involved in defined events in cardio-genesis are being uncovered at a rapid rate. Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. (4, 5, 6). However, our ability to cross the divide between knowledge and change has not been easy, as reported by Aviva Lev-Ari (7). In a two-day-old mouse, a heart attack causes active stem cells to grow new heart cells; a few months later, the heart is mostly repaired. But in an adult mouse, recovery from such an attack leads to classic after-effects: scar tissue, permanent loss of function and life-threatening arrhythmias (7, 8).

Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The success using dermal fibroblasts from HF patients reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector resulted in HF-hiPSCs induced to differentiate into cardiomyocytes (HF-hiPSC-CMs)(9). Multi-electrode array recordings revealed adequate responses to stimulation. Further study with in vivo transplantation in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes and within 48 hours the tissues were beating together. Human-induced pluripotent stem cells thus can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue (10). The success of the approach rests on modifying the myocardial electro-physiological substrate using cell grafts genetically engineered to express specific ionic channels (11). The expressed potassium channels alter the local myocardial electrophysiological properties by reducing cardiac automaticity and prolonging refractoriness. The key feature involves reprogramming a patient’s own skin cells by delivering three genes followed by a small molecule called valproic acid to the cell nucleus (12).

An alternative approach avoiding the caveats of limited graft survival, is to stimulate a resident source, restricted homing to the site of injury and host immune rejection (13). Thymosin β4 restores vascular potential to adult epicardial-derived progenitor cells with injury. Specifically, it activates adult progenitors to re-express a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1). It was inferred that embryonic reprogramming would mobilize this cell population and differentiation would give rise to de novo cardiomyocytes. Delivery of Tβ4, in conjunction with GMT (an acronym for three genes that normally guide embryonic heart development), into the damaged region resulted in reduction of scar area and improvement in cardiac function compared to GMT or Tβ4 alone. Thymosin-beta4 facilitates cardiac repair after infarction by promoting cell migration and myocyte survival. Additionally, the tetra peptide Ac-SDKP was reported to reduce left ventricular fibrosis in hypertensive rats, reverse fibrosis and inflammation in rats with MI, and stimulate both in vitro and in vivo angiogenesis. Effects of Ac-SDKP, such as the enhancement of angiogenesis and the decrease in inflammation and collagenase activity, are similar to those described for thymosin-beta4. However, there are conflicting reports (14-18).

There are other studies that show promise. There has been the first infusion of stem cells into the coronary artery (19). This result was at least as effective as intramyocardial injection in limiting LV remodeling and improving both regional and global LV function. The intracoronary route appears to be superior in terms of uniformity of cell distribution, myocyte regeneration, and amount of viable tissue in the risk region. Another finds that down regulation of leukocyte HIF-1? Expression resulted in decreased recruitment of WBC to the sites of inflammation and improvement in cardiac function following MI (20). Irradiated 6-to 8-week-old C57/BL6J mice received 50 000 GFP HIF-1? or scramble siRNA transfected hematopoietic stem cells. Down regulation of HIF-1? suppressed WBC cytokine receptors CCR1,-2, and-4, which are necessary for WBC mobilization and recruitment to inflammatory cytokines following MI. There also have been cited limitations to success in older patients (21). The findings suggest that coronary artery disease and cardiac remodeling in chronic ischemia has a significant negative correlation between the age of the patient and the number of migrated ckit-positive cells.

Lymphocytes infiltrate and react with ischemia damaged heart tissue, which can impair proper tissue healing. In a study with isoproterenol induced myocardial necrosis TNF-α, IFN-γ and CCL-5, but not FOXP3 + expression, was increased in draining lymph nodes, indicating that the observed lymphocyte population that proliferated in response to cardiac components presented a pro-inflammatory and pro-fibrotic profile. The group was rendered tolerant by myocardial gavage and expressed cardiac FOXP3 + earlier than did the control group, and showed a milder inflammatory infiltrate, lower MMP-9 expression, less collagen deposition, and improved cardiac performance when compared to animals that received only isoproterenol administration (22). Patients with acute myocardial infarction show high circulating levels of neuropeptide Substance P (SP) and NK1-positive cells that co express Progenitor Cell (PC) antigen, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC is abundant in infarcted hearts, highlighting the role of SP in reparative neovascularization (23). Do CD4 + T cells become activated and influence wound healing after experimental MI? To study the role of CD4 + T cells in wound healing and remodeling, CD4 + T-cell- deficient mice (CD4 knockout [KO], MHCII) and T-cell receptor-transgenic OT-II. Within the infarcted myocardium, CD4 KO mice displayed higher total numbers of leukocytes and proinflammatory monocytes (18.3±3.0 104/mg WT versus 75.7±17.0 10 4/mg CD4 KO, P<0.05), and MHCII and OT-II mice displayed significantly greater mortality. Collagen matrix formation in the infarct zone was severely disturbed in CD4 KO and MHCII mice, as well as in OT-II mice (24).

Thus, it appears that CD4T cells become activated after MI and facilitate wound healing of the myocardium. Inflammation and immune responses are integral components in he healing process after myocardial infarction. Importantly, dendritic cell (DC) infiltration occurs in the infarcted heart. In concert with the previous two studies, DC-ablated infarcts had enhanced monocyte/ macrophage recruitment. Among these cells, marked infiltration of proinflammatory Ly6C high monocytes and F4/80 + CD206 – M1 macrophages and, conversely, impaired recruitment of anti-inflammatory Ly6C low monocytes and F4/80 + CD206 + M2 macrophages in the infarcted myocardium were identified in the DC-ablated group compared with the control group (25). Thus, the DC is a potent immunoprotective regulator during the post-infarction healing process via its control of monocyte/macrophage homeostasis. Despite the recent successes, there are a number of interlocking and possibly explanatory processes to control in the mix.

What about medical therapies? Here too there is a factor in engaging eNOS or iNOS activity as detailed in the presentation by Aviva Lev-Ari (26). 60–70% of major cardiovascular events cannot be prevented with current approaches focused on LDL, such as statin therapy, and low HDL levels are particularly common in males with early-onset atherosclerosis. She makes the point that there is compelling evidence that HDL is not solely a marker of lower risk of cardiovascular disease but instead is a mediator of vascular health.

Aviva Lev-Ari examines the phytoestrogen, Genistein, and other drugs. Genistein acutely stimulates Nitric Oxide synthesis in vascular Endothelial cells by a cyclic adenosine 5′-monophosphate-dependent mechanism (Liu et al., 2004). The intracellular signaling pathways for activation of eNOS by genistein were shown independent of PI3K/Akt or ERK/MAPK but depended on the cAMP/PKA cascade. In addition, the genistein action on eNOS was not inhibited by an ER antagonist and was unrelated to tyrosine kinase inhibition. Furthermore, genistein has antiatherogenic effects and inhibits proliferation of vascular endothelial and smooth muscle cells, and in vitro studies suggest a protective role of genistein in the vasculature. In Liu et al., (2004) study, genistein acted directly on BAECs and HUVECs to activate eNOS and NO production through nongenomic mechanisms in whole vascular endothelial cells. In addition, 5-hydroxytryptamine evokes endothelial nitric oxide synthase activation. In this example, eNOS co-localizes with PECAM-1, but not with VE-cadherin and plakoglobin at the intercellular junctions of the endothelium.

Finally, activation of endothelial nitric oxide synthase (eNOS) resulted in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The responses were effectively blocked by a 5-HT1B receptor antagonist, a 5-HT1B/5-HT2 receptor antagonist, and eNOS selective antagonists, L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). This lends credence to a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Finally, a third-generation ß-blocker augments vascular Nitric Oxide release. Nebivolol increases vascular NO productionby causing a rise in endothelial free [Ca2+]i and endothelial NO synthase–dependent NO production. It is a ß1-selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties. Nevertheless, it appears that not nebivolol, but its metabolites augment NO production (Broeders et al., 2000). These findings reveal new insights into interaction with eNOS in vascular therapy: [1] new indications for TDZs as stimulators of eNOS; [2] new indications for beta blockers as NO stimulant. Nebivolol is a vasodilator, thus functions as an antihypertensive.

References:

1. Saha S. Innovations in Bio-instrumentation for Measurement of Circulating Progenitor Endothelial Cells in Human Blood. Pharma Intell. July 8, 2012. http://pharmaceuticalintelligence.com/2012/07/08/innovations-in-bio-instrumentation-for-measurement-of-circulating-progenitor-endothelial-cells-in-human-blood/

(http://www.ncbi.nlm.nih.gov/pubmed/19124422 )

2. Ibid (http://www.ncbi.nlm.nih.gov/pubmed/20381496).

3. Ibid (http://zh.invitrogen.com/etc/medialib/files/Cell-Analysis/PDFs.Par.54318.File.tmp/CO24210-Human-CEC_cancer.pdf)

4. Saha S. Endothelial Differentiation and Morphogenesis of Cardiac Precursors. Pharma Intelligence. July 17, 2012.

5. Ibid (http://circres.ahajournals.org/content/90/5/509.full).

6. Ibid (http://www.ncbi.nlm.nih.gov/pubmed/22669846).

7. Aviva-Lev-Ari. Stem cells create new heart cells in baby mice, but not in adults, study shows.Aug 3, 2012. Pharma Intelligence.

8. Krishna Ramanujan http://www.news.cornell.edu/stories/July12/HeartStemCells.html

9. Saha S. Human Embryonic-Derived Cardiac Progenitor Cells for Myocardial Repair. Pharma Intelligence. Aug 1, 2012.

10. Zwi-Dantsis L, Huber I, Habib M, Winterstern A, (..), Gepstein L. Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients. Eur Heart J. 2012 May 22. [Epub ahead of print] (VBL RX, Inc. Tel Aviv, http://www.vblrx.com).

11. Yankelson L, Feld Y, Bressler-Stramer T, Itzhaki I,(..), Gepstein L. Cell therapy for modification of the myocardial electrophysiological substrate. Circulation. 2008 Feb 12; 117(6):720-31. Epub 2008 Jan 22.

12. Huber I, Itzhaki I, Caspi O, Arbel G, (..), Gepstein L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J. 2007 Aug; 21(10):2551-63. Epub 2007 Apr 13.

13. Aviva Lev-Ari. Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair. Pharma Intelligence. April 30, 2012.

14. Ibid. Shrivastava S, Srivastava D, Olson EN, DiMaio JM, Bock-Marquette I.

Ann N Y Acad Sci. 2010 Apr; 1194:87-96.

15. Ibid. Smart N, Risebro CA, Clark JE, Ehler E, (..), Riley PR, Thymosin beta4 facilitates epicardial neovascularization of the injured adult heart. Ann N Y Acad Sci. 2010 Apr;1194:97-104

16. Ibid. Smart N, Bollini S, Dubé KN, Vieira JM, (..) Riley PR. Nature. 2011 Jun 8; 474(7353):640-4

17. Ibid. Zhou B, Honor LB, Ma Q, Oh JH, (..), Pu WT. Thymosin beta 4 treatment after myocardial infarction does not reprogram epicardial cells into cardiomyocytes. J Mol Cell Cardiol. 2012 Jan; 52(1):43-7. Epub 2011 Aug 26.

18. Ibid. Scientists Report that Process of Converting Non-Beating Heart Cells into Functional, Beating Heart Cells is Enhanced Using Thymosin Beta 4 in Conjunction with Gene Therapy. Regenerx Biopharmaceuticals, Inc. Nature. Apr. 18, 2012

19. Li, Q., Guo, Y., Ou, Q., Chen, N., (…), Bolli, R. Intracoronary administration of cardiac stem cells in mice: A new, improved technique for cell therapy in murine models. Basic Research in Cardiology 2011; 106 (5), pp. 849-864.

20. Dong, F., Khalil, M.,Kiedrowski, M.,O’Connor, C.,(..) ,Penn, M.S. Critical role for leukocyte hypoxia inducible factor-1α expression in post-myocardial infarction left ventricular remodeling. Circulation Research 2010; 106 (3) , pp. 601-610

21. Aghila Rani, K.G.,Jayakumar, K.,Sarma, P.S., Kartha, C.C. Clinical determinants of ckit-positive cardiac cell yield in coronary disease. Asian Cardiovascular and Thoracic Annals 2009; 17 (2), pp. 139-142.

22. Ramos, G.C., Dalbó, S., Leite, D.P.,Goldfeder, E. ,(..), Assreuy, J. The autoimmune nature of post-infarct myocardial healing: Oral tolerance to cardiac antigens as a novel strategy to improve cardiac healing. Autoimmunity 2012; 45 (3), pp. 233-244.

23. Amadesi, S., Reni, C., Katare, R., Meloni, M., (…),Madeddu, P. Role for substance P-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects. Circulation 2012; 125 (14) , pp. 1774-1786.

24. Hofmann, U.,Beyersdorf, N.,Weirather, J.,Podolskaya, A.(..), Frantz, S. Activation of CD4 + T lymphocytes improves wound healing and survival after experimental myocardial infarction in mice. Circulation 2012; 125 (13) , pp. 1652-1663.

25. Anzai, A., Anzai, T.,Nagai, S., Maekawa, Y., (…), Fukuda, K. Regulatory role of dendritic cells in postinfarction healing and left ventricular remodeling. Circulation 2012; 125 (10), pp. 1234-1245

26. Lev-Ari A. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production. Pharma Intelligence. July 19, 2012.

27. Ibid. Li AC, Binder CJ, Gutierrez A, Brown KK, (..), Glass CK. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPAR-alpha, Beta/delta, and gamma. J Clin Invest 2004; 114:1564-1576.

28. Ibid. Broeders MAW, Doevendans PA, Bekkers BCAM, (…), van der Zee R. Nebivolol: A Third-Generation ß-Blocker That Augments Vascular Nitric Oxide Release, Endothelial ß2-Adrenergic Receptor–Mediated Nitric Oxide Production. Circulation 2000; 102:677.