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Li -Fraumeni Syndrome and Pancreatic Cancer

Posted in Cancer Screening, Cell Biology, Genome Biology, Genomic Testing: Methodology for Diagnosis, Li-fraumeni syndrome., Mutagenesis, TP53 - Germline mutations, Variation in human protein-coding regions, tagged gene therapy, Li-fraumeni syndrome., Pancreatic cancer, PRIMA-1, TP53 on December 14, 2016| Leave a Comment »

Li -Fraumeni Syndrome and Pancreatic Cancer

Curator: Marzan Khan, B.Sc.

Li-Fraumeni syndrome (LFS) is a condition that makes individuals prone to developing a wide variety of cancers that occur early on in life, the most common types being- soft tissue sarcoma, osteosarcoma, breast cancer, brain tumors, adrenocortical carcinoma (ACC), and leukemia. (1) Pancreatic cancer is minimally associated with the condition. (2) A survey found the presence of pancreatic cancer in only 1% of 475 tumor samples collected from 91 families who were carriers of p53 mutations, with half of them having LFS. The incidence of breast cancer amongst them was the highest -24%. (2) Pancreatic carcinoma in LFS patients usually occurs in the later stages of life. (3)

The underlying cause of LFS is germline mutations in TP53 gene on chromosome 17p, that encodes the transcription factor p53, crucial in cell cycle regulation and the repair of damaged and/or abnormal cells. (4) In the majority of cases, this mutation is obtained by inheritance. (5) De-novo germline mutations in p53 occur in 7%-20% of the cases. (5)

A person showing symptoms of any type of cancer at an early age or having first or second-degree relatives with cancer are at risk of developing LFS. (5) That is why tracing family history is an important part of diagnosis in LFS patients. Genetic testing can confirm mutations present in the gene, however, there are controversial ethical issues regarding their use, particularly in children and fetuses.

In patients with LFS, it is important to control the manifestations of the disease. They should be monitored closely so that any new cancers that arise are diagnosed and treated during the early stages. (6) Patients are also at risk of developing radiation-induced second and third primary tumors. (6) Therefore, radiation and alkylating agents should be used minimally (6) People at risk can be cautioned to avoid exposure to carcinogens such as sunlight, cigarette smoke, and alcohol consumption. (5) Therapeutic approaches that are aimed at restoring wild-type p53 by gene therapy as well as reactivating non-functional p53 by the use of small-molecule drugs are currently being investigated in many cancers. (7) Unlike radiation therapy, these small-molecule drugs are non-toxic to healthy cells, thus eliminating the risk of forming new tumors.

So far, PRIMA-1 has proven to be quite effective at correcting non-functional p53. (8) PRIMA-1 is changed to its methylated form, PRIMA-1^METthat forms covalent adducts to thiol groups in the mutated protein and modifies them. (8) As a result, p53 regains its ability to destroy malignant cells. (8) A research study also found that PRIMA-1 induces apoptosis and increases the sensitivity of pancreatic cancer cells to various chemotherapeutic agents. (9)

Magali Olivier, David E. Goldgar, Nayanta Sodha, Hiroko Ohgaki, Paul Kleihues, Pierre Hainaut and Rosalind A. Eeles. Li-Fraumeni and Related Syndromes. Cancer Res October 15 2003 63 (20) 6643-6650 http://cancerres.aacrjournals.org/content/63/20/6643.abstract
Kleihues P, Schauble B, zur Hausen H, et al. Tumors associated with p53 germline mutations: a synopsis of 91 families. Am J Pathol 1997; 150:1-13 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1858532/
John P. Neoptolemos, Raul Urrutia, James L. Abbruzzese, Markus W. Buchler. Pancreatic Cancer. 2010.1^st ed, pp-6, 2010, Springer, Verlag, New York
Mishra B and Patel RR. Gene Therapy for Treatment of Pancreatic Cancer. Austin Therapeutics. 2014;1(1): 10. https://books.google.ca/books?id=NmBB5ZoKkk4C&pg=PA6&lpg=PA6&dq=connection+between+li+fraumeni+and+Pancreatic+cancer&source=bl&ots=H0iCeaPP0N&sig=pqJT1tPMR6C-NIig3S_NkFKFsD0&hl=en&sa=X&ved=0ahUKEwi4nLrgzuPQAhUUIWMKHS3wBoc4ChDoAQhNMAg#v=onepage&q=connection%20between%20li%20fraumeni%20and%20Pancreatic%20cancer&f=false
Schneider K, Zelley K, Nichols KE, et al. Li-Fraumeni Syndrome. 1999 Jan 19 [Updated 2013 Apr 11]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. https://www.ncbi.nlm.nih.gov/pubmed/20301488
Elisa Becze BA, ELS, 2011 Mar 1. An introduction to Li-Fraumeni Syndrome, Five-Minute-In-Service. http://connect.ons.org/columns/five-minute-in-service/an-introduction-to-li-fraumeni-syndrome
Sorrell, A. D., Espenschied, C. R., Culver, J. O., & Weitzel, J. N. (2013).TP53Testing and Li-Fraumeni Syndrome: Current Status of Clinical Applications and Future Directions. Molecular Diagnosis & Therapy, 17(1), 31–47. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627545/
Emily J. Lewis. PRIMA-1 as a cancer therapy restoring mutant p53: a reviewBioscience Horizons (2015) 8: hzv006 http://biohorizons.oxfordjournals.org/content/8/hzv006.full
Izetti, Patricia, Agnes Hautefeuille, Ana Lucia Abujamra, Caroline Brunetto de Farias, Juliana Giacomazzi, Bárbara Alemar, Guido Lenz, et al. ‘PRIMA-1, a Mutant p53 Reactivator, Induces Apoptosis and Enhances Chemotherapeutic Cytotoxicity in Pancreatic Cancer Cell Lines’. Investigational New Drugs 32, no. 5 (October 2014): 783–94. https://www.ncbi.nlm.nih.gov/pubmed/24838627

Izetti, Patricia, Agnes Hautefeuille, Ana Lucia Abujamra, Caroline Brunetto de Farias, Juliana Giacomazzi, Bárbara Alemar, Guido Lenz, et al. ‘PRIMA-1, a Mutant p53 Reactivator, Induces Apoptosis and Enhances Chemotherapeutic Cytotoxicity in Pancreatic Cancer Cell Lines’. Investigational New Drugs 32, no. 5 (October 2014): 783–94

Mid Atlantic LRIG 22nd Annual Technology Showcase: Agenda on 3D Bioprinting on Wednesday, May 11, 2016; Somerset NJ

Posted in 3D Plotting Scaffolds, 3D Printing for Medical Application, Drug Delivery Platform Technology, tagged 3D bioprinting, advances in drug development, Cell therapy, Clinical Laboratory Automation, gene therapy, scientific meeting on April 16, 2016| Leave a Comment »

Mid Atlantic LRIG 22nd Annual Technology Showcase: Agenda on 3D Bioprinting on Wednesday, May 11, 2016 at Holiday Inn, 195 Davidson Avenue, Somerset, NJ

Reporter: Stephen J. Williams, Ph.D.

Symposium Speakers and Topics:

Human Organoids
Hatem E. Sabaawy-Director, Production GMP Facility for Cell and Gene Therapy, RBHS-Robert Wood Johnson Medical School, Rutgers Cancer Institute of New Jersey

Intestinal Organoids for Drug Discovery
Richard Visconti-Associate Principal Scientist, Cellular Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey

3D Bioprinting
Elizabeth Wu-President, WuZenTech, Edison, New Jersey

Building Your Brand Through LinkedIn
Stan Robinson, Jr., LinkedIn Consultant, Helping Professionals with Social Selling, Personal Branding

Register at EventBrite here: https://www.eventbrite.com/e/mid-atlantic-22nd-annual-technology-and-exhibition-tickets-21359945171

To sign up to be an LRIG member or update your profile, please visit us at http://lrig.org
Hoping to see you on May 11th.
R eserve your spot today!

AGTC (AGTC) , An adenoviral gene therapy startup, expands in Florida with help from $1 billion deal with Biogen

Posted in Genetics & Innovations in Treatment, Genome Biology, Investment in Technological Breakthrough, Medical Devices R&D Investment, tagged AAV, adenovirus, biotech startup, gene therapy, genetic diseases, Global Partnering & Biotech Investment, startup, Venture capital on February 17, 2016| Leave a Comment »

AGTC (AGTC) , An adenoviral gene therapy startup, expands in Florida with help from $1 billion deal with Biogen

Reporter: Stephen J. Williams, Ph.D.

from Biospace News

AGTC Sets Up Shop in Florida, New Facility to House 75 Employees
February 17, 2016
By Alex Keown, BioSpace.com Breaking News Staff

GAINESVILLE, Fla. — Applied Genetic Technologies Corporation (AGTC), a biotechnology company researching adeno-associated virus (AAV)-based gene therapies for the treatment of rare diseases, is expanding into the rapidly growing north central Florida biotech corridor.

The company, which was founded on technology developed at the University of Florida, is opening a combined use corporate office and laboratory facility in Alachua, Fla. AGTC’s portion of the new multi-tenant facility is expected to accommodate up to about 75 people and consists of approximately 20,000 square feet including state-of-the-art lab and office space as well as space for future expansion, the company announced this morning.

“The new facility will help us to accelerate our research and development efforts for novel AAV-based gene therapies for rare diseases and house critical corporate functions including finance, quality assurance and project management, while providing ample space as we continue to bring new talent to our team,” Sue Washer, president and chief executive officer of AGTC said in a statement.

AGTC’s lead product candidates focus on X-linked retinoschisis, achromatopsia and X-linked retinitis pigmentosa, which are inherited orphan diseases of the eye, caused by mutations in single genes that significantly affect visual function and currently lack effective medical treatments. Retinoschisis is a condition in which an area of the retina has separated into two layers. The part of the retina that is affected by retinoschisis will have suboptimal vision, according to the University of Michigan’s Kellogg Eye Center. Achromatopsia is a condition of the eye that is characterized by an absence (partial or total) of color vision. People with the complete form of achromatopsia are unable to perceive any colors and can only see black, white and shades of gray.

AGTC is also pursuing pre-clinical development of treatments for wet AMD using the company’s experience in ophthalmology to expand into disease indications with larger markets.

In August, AGTC’s research was bolstered by a $1 billion deal withBiogen (BIIB) to support the company’s gene-based therapies. As part of the deal, Biogen holds a license to AGTC’s XLRS and XLRP programs and an additional three licenses, BioSpace (DHX) reported in August.

David Day, assistant vice president & director of the Office of Technology Licensing at the University of Florida, touted the growth of the biotech sector in north central Florida.

“AGTC’s progress in developing novel treatments for rare diseases without adequate therapeutic options is a particularly good model for the entire biotechnology sector,” Day said in a statement.

FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials

Posted in 3D Printing for Medical Application, CRISPR/Cas9 & Gene Editing, FDA, FDA Regulatory Affairs, Tissue Engineering, tagged Clinical trial, CRISPR-Cas9, Editas, FDA, FDA guidance, Food and Drug Administration, gene, gene expression, gene therapy, genetics, medicine on November 10, 2015| Leave a Comment »

FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials

Reporter: Stephen J. Williams, PhD

The recent announcement by Editas CEO Katrine Bosley to pursue a CRSPR/Cas9 gene therapy trial to correct defects in an yet to be disclosed gene to treat one form of a rare eye disease called Leber congenital amaurosis (multiple mutant genes have been linked to the disease) have put an interesting emphasis on the need for a regulatory framework to initiate these trials. Indeed at the 2015 EmTechMIT Conference Editas CEO Katrine Bosley had mentioned this particular issue: the need for discourse with FDA and regulatory bodies to establish guidelines for design of clinical trials using the CRSPR gene editing tool.

See the LIVE NOTES from Editas CEO Katrine Bosley on using CRSPR as a gene therapy from the 2015 EmTechMIT Conference at http://pharmaceuticalintelligence.com/2015/11/03/live-1132015-130pm-the-15th-annual-emtech-mit-mit-media-lab-top-10-breakthrough-technologies-2015-innovators-under-35/

To this effect, I have listed below, the multiple FDA Guidance Documents surrounding gene therapy to show that, in the past year, the FDA has shown great commitment to devise a regulatory framework for this therapeutic area.

Cellular & Gene Therapy Guidance Documents

Homologous Use of Human Cells, Tissue, and Cellular and Tissue-Based Products; Draft guidance for Industry and FDA Staff (PDF – 120KB)
10/2015
Recommendations for Microbial Vectors Used for Gene Therapy; Draft Guidance for Industry (PDF – 447KB)
10/2015
Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products; Guidance for Industry (PDF – 120KB)
8/2015
Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products; Guidance for Industry (PDF – 313KB)
6/2015
Determining the Need for and Content of Environmental Assessments for Gene Therapies, Vectored Vaccines, and Related Recombinant Viral or Microbial Products; Guidance for Industry
3/2015
Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) from Adipose Tissue: Regulatory Considerations; Draft Guidance for Industry
12/2014. For more information, please refer to footnote 1 in the 2015 GUIDANCE AGENDA under RESOURCES FOR YOU.
Minimal Manipulation of Human Cells, Tissues, and Cellular and Tissue-Based Products: Draft Guidance for Industry and Food and Drug Administration Staff
12/2014. When finalized, this guidance will supersede the FDA guidance entitled, “Minimal Manipulation of Structural Tissue Jurisdictional Update,” September 2006. Please refer to footnote 1 in the 2015 GUIDANCE AGENDA under RESOURCES FOR YOU.
Draft Guidance for Industry: Same Surgical Procedure Exception under 21 CFR 1271.15(b): Questions and Answers Regarding the Scope of the Exception
10/2014. For more information, please refer to footnote 1 in the 2015 GUIDANCE AGENDA under RESOURCES FOR YOU.
Guidance for Industry: BLA for Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic and Immunologic Reconstitution in Patients with Disorders Affecting the Hematopoietic System (PDF – 385KB)
3/2014. (This guidance finalizes the draft guidance of the same title dated June 2013.)
Guidance for Industry and FDA Staff: IND Applications for Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic and Immunologic Reconstitution in Patients with Disorders Affecting the Hematopoietic System
3/2014. (This guidance finalizes the draft guidance of the same title dated June 2013.)
Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products
November 2013. (This guidance finalizes the draft guidance entitled “Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products” dated November 2012)
Guidance for Industry: Preparation of IDEs and INDs for Products Intended to Repair or Replace Knee Cartilage (PDF – 157KB)
12/2011. (This guidance finalizes the draft guidance of the same title dated July 2007.)
Guidance for Industry: Clinical Considerations for Therapeutic Cancer Vaccines (PDF – 75KB)
10/2011. (This guidance finalizes the draft guidance of the same title dated September 2009.)
Guidance for Industry and FDA Staff: Investigational New Drug Applications for Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic Reconstitution for Specified Indications. (PDF – 104KB)
6/2011
Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (PDF – 311KB)
1/2011. (This guidance finalizes the draft document of the same name, dated October 2008.)
Guidance for Industry: Cellular Therapy for Cardiac Disease
10/2010. (This guidance finalizes the draft guidance entitled “Guidance for Industry: Somatic Cell Therapy for Cardiac Disease” dated March 2009 (April 2, 2009, 74 FR 14992).
Draft Guidance for Industry: Assay Development for Immunogenicity Testing of Therapeutic Proteins (PDF – 161KB)
12/2009
Guidance for Industry – Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic Reconstitution for Specified Indications (PDF – 462KB)
10/2009
Guidance for Industry: Considerations for Allogeneic Pancreatic Islet Cell Products
9/2009
Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)
4/2008
Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs)
4/2008
Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products
8/2007
Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events
11/2006
Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors
11/2006
Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy
3/1998

Withdrawn Guidance Documents

Withdrawn – Draft Guidance for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products
May 2015

Three other posts on this site goes into detail into three of the above-mentioned Guidance Documents

FDA Guidance on Use of Xenotransplanted Products in Human: Implications in 3D Printing

New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

FDA Guidance Documents Update Nov. 2015 on Devices, Animal Studies, Gene Therapy, Liposomes

10/14/15: General Considerations for Animal Studies for Medical Devices – Draft Guidance for Industry and Food and Drug Administration Staff

FDA Guidance Documents Update Nov. 2015 on Devices, Animal Studies, Gene Therapy, Liposomes

Posted in FDA, FDA Regulatory Affairs, FDA, CE Mark & Global Regulatory Affairs: process management and strategic planning - GCP, GLP, ISO 14155, Lipidomics, Liposome, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, Uncategorized, tagged animal testing, Clinical trial, FDA, Food and Drug Administration, gene therapy, health on November 3, 2015| Leave a Comment »

FDA Guidance Documents Update

Reporter: Stephen J. Williams, Ph.D.

10/30/2015

You are subscribed to FDA Guidance Documents for U.S. Food & Drug Administration (FDA).

This information has recently been updated and is now available.

Recently posted guidance documents

10/14/15: Recommendations for Microbial Vectors Used for Gene Therapy; Draft Guidance for Industry

10/15/15: Draft PDEs for Triethylamine and for Methylisobutylketone

10/15/15: ICH Q3C Maintenance Procedures for the Guidance for Industry Q3C Impurities: Residual Solvents

10/19/15: CVM GFI #229 – Evaluating the Effectiveness of New Animal Drugs for the Reduction of Pathogenic Shiga Toxin-Producing E. coli in Cattle

10/21/15: Selection of the Appropriate Package Type Terms and Recommendations for Labeling Injectable Medical Products Packaged in Multiple-Dose, Single-Dose, and Single-Patient-Use Containers for Human Use

10/21/15: Manufacturing Site Change Supplements: Content and Submission – Draft Guidance for Industry and Food and Drug Administration Staff

10/26/15: Interim Policy on Compounding Using Bulk Drug Substances Under Section 503A of the Federal Food, Drug, and Cosmetic Act Guidance for Industry

10/26/15: Interim Policy on Compounding Using Bulk Drug Substances Under Section 503B of the Federal Food, Drug, and Cosmetic Act

10/26/15: Pharmacy Compounding of Human Drug Products Under Section 503A of the Federal Food, Drug, and Cosmetic Act Guidance

10/27/15: Nonclinical Safety Evaluation of Reformulated Drug Products and Products Intended for Administration by an Alternate Route

10/27/15: Product Development Under the Animal Rule

10/28/15: DSCSA Implementation: Product Tracing Requirements for Dispensers — Compliance Policy (Revised) Guidance for Industry

10/29/15: Liposome Drug Products: Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation

Guidance Document Search

• Search all FDA official guidance documents and other regulatory guidance

Source: https://www.fiercepharma.com/ai-and-machine-learning/oncologists-have-shopping-car-t-full-complaints-safety-questions-cell?utm_medium=email&utm_source=nl&utm_campaign=LS-NL-FiercePharma+Tracker&oly_enc_id=2360C5096034F3G

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

Posted in Autologous Cell Therapy, Bioengineering & reverse engineering design, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, CE Mark & Global Regulatory Affairs: process management and strategic planning - GCP, Computational Biology/Systems and Bioinformatics, Disease Biology, Drug Toxicity, FDA, FDA Regulatory Affairs, Genetics & Pharmaceutical, GLP, Human Immune System in Health and in Disease, Immunodiagnostics, Innovation in Immunology Diagnostics, Innovations, ISO 14155, Personalized and Precision Medicine & Genomic Research, Quality Assurance, Translational Research, tagged Adult ALL, Adverse event, Alliance for Clinical Trials in Oncology, Apoptosis, autologous transplant, B and T cell gene rearrangements, Cancer - General, CD8+ T cells, Clinical trial, clinical trial guidelines, Food and Drug Administration, gene expression, gene therapy, health, insertional mutagenesis, interleukins, medicine, National Institutes of Health, Personalized medicine, Pharmacovigilance, research, safety guidelines on September 23, 2014| Leave a Comment »

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

Reporter: Stephen J. Williams, Ph.D.

UPDATED 5/27/2024

The practice of pharmacovigilence, both premarketing and postmarketing, has very well defined best practices concerning most small molecule drugs and even medical devices. However, for many cell based therapies and many gene based therapies, often still administered within the university, academic setting, pharmacovigilence reporting and adherence may be a not as efficient and thorough as conducted by large big pharmaceutical firms. Big pharma will devote massive resources for the conduct of pharmacovigilence data collecting and analysis. For many cell based therapies, like CAR-T therapies and some gene therapies are almost conducted as clinical trials within university medical centers, which may not have the resources for a large pharmacovigilence program.

In a report by IQVIA, oncologists were asked about their concerns with cell based therapies. A recurring concern involved the lack of information on the adverse events related to these therapies, especially after an oncologist’s patient would return from administration of their CAR-T therapy and then both patient and oncologist felt ‘on their own’.

Most recently the FDA has issued black box warning on many CAR-T therapies for their risk in inducing secondary malignancies (see What does this mean for Immunotherapy? FDA put a temporary hold on Juno’s JCAR015, Three Death of Cerebral Edema in CAR-T Clinical Trial and Kite Pharma announced Phase II portion of its CAR-T ZUMA-1 trial).

Note: the IQVIA will be submitted as an abstract at the current ASCO meeting

UPDATED 5/10/2022

In the mid to late 1970’s a public debate (and related hysteria) had emerged surrounding two emerging advances in recombinant DNA technology;

the development of vectors useful for cloning pieces of DNA (the first vector named pBR322) and
the discovery of bacterial strains useful in propagating such vectors

As discussed by D. S, Fredrickson of NIH’s Dept. of Education and Welfare in his historical review” A HISTORY OF THE RECOMBINANT DNA GUIDELINES IN THE UNITED STATES” this international concern of the biological safety issues of this new molecular biology tool led the National Institute of Health to coordinate a committee (the NIH Recombinant DNA Advisory Committee) to develop guidelines for the ethical use, safe development, and safe handling of such vectors and host bacterium. The first conversations started in 1974 and, by 1978, initial guidelines had been developed. In fact, as Dr. Fredrickson notes, public relief was voiced even by religious organizations (who had the greatest ethical concerns)

On December 16, 1978, a telegram purporting to be from the Vatican was hand delivered to the office of Joseph A. Califano, Jr., Secretary of Health, Education,

and Welfare. “Habemus regimen recombinatum,” it proclaimed, in celebration of the

end of a long struggle to revise the NIH Guidelines for Research Involving

Recombinant DNA Molecules

The overall Committee resulted in guidelines (2013 version) which assured the worldwide community that

organisms used in such procedures would have limited pathogenicity in humans
vectors would be developed in a manner which would eliminate their ability to replicate in humans and have defined antibiotic sensitivity

So great was the success and acceptance of this committee and guidelines, the NIH felt the Recombinant DNA Advisory Committee should meet regularly to discuss and develop ethical guidelines and clinical regulations concerning DNA-based therapeutics and technologies.

A PowerPoint Slideshow: Introduction to NIH OBA and the History of Recombinant DNA Oversight can be viewed at the following link:

http://www.powershow.com/view1/e1703-ZDc1Z/Introduction_to_NIH_OBA_and_the_History_of_Recombinant_DNA_Oversight_powerpoint_ppt_presentation

Please see the following link for a video discussion between Dr. Paul Berg, who pioneered DNA recombinant technology, and Dr. James Watson (Commemorating 50 Years of DNA Science):

http://media.hhmi.org/interviews/berg_watson.html

The Recombinant DNA Advisory Committee has met numerous times to discuss new DNA-based technologies and their biosafety and clinical implication including:

January 24, 2013 Biosafety Considerations for Research with Highly Pathogenic Avian Influenza Virus H5N1 that is Transmissible between Mammals by Respiratory Droplets

2012 Discussion of Gene Transfer Protocols

2008 Guidelines for HIV Vaccination Protocols Appendix M-VI-A of the NIH Guidelines (the “Vaccine Exemption”)

2006 Guidance on Biosafety Considerations for Research with Lentiviral Vectors

A recent Symposium was held in the summer of 2010 to discuss ethical and safety concerns and discuss potential clinical guidelines for use of an emerging immunotherapy technology, the Chimeric Antigen Receptor T-Cells (CART), which at that time had just been started to be used in clinical trials.

Considerations for the Clinical Application of Chimeric Antigen Receptor T Cells: Observations from a Recombinant DNA Advisory Committee Symposium Held June 15, 2010[1]

Contributors to the Symposium discussing opinions regarding CAR-T protocol design included some of the prominent members in the field including:

Drs. Hildegund C.J. Ertl, John Zaia, Steven A. Rosenberg, Carl H. June, Gianpietro Dotti, Jeffrey Kahn, Laurence J. N. Cooper, Jacqueline Corrigan-Curay, And Scott E. Strome.

The discussions from the Symposium, reported in Cancer Research[1]. were presented in three parts:

Summary of the Evolution of the CAR therapy
Points for Future Consideration including adverse event reporting
Considerations for Design and Implementation of Trials including mitigating toxicities and risks

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

However there were some problems noticed.

Problem: HLA restriction of T-cells. Solution: genetically engineer T-cells to redirect T-cell specificity to surface TAAs
Problem: 1^st generation vectors designed to engineer T-cells to recognize surface epitopes but engineered cells had limited survival in patients. Solution: development of 2^nd generation vectors with co-stimulatory molecules such as CD28, CD19 to improve survival and proliferation in patients

A summary table of limitations of the two types of genetically-modified T-cell therapies were given and given (in modified form) below

Type of Gene-modified T-Cell

Limitations	aβ TCR	CAR
Affected by loss or decrease of HLA on tumor cells	yes	no
Affected by altered tumor cell antigen processing?	yes	no
Need to have defined tumor target antigen?	no	yes
Vector recombination with endogenous TCR	yes	no

A brief history of construction of 2^nd and 3^rd generation CAR-T cells given by cancer.gov:

http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells

Differences between second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from cancer.gov)

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Adverse Event Reporting and Protocol Considerations

The symposium had been organized mainly in response to two reported deaths of patients enrolled in a CART trial, so that clinical investigators could discuss and formulate best practices for the proper conduct and analysis of such trials. One issue raised was lack of pharmacovigilence procedures (adverse event reporting). Although no pharmacovigilence procedures (either intra or inter-institutional) were devised from meeting proceedings, it was stressed that each institution should address this issue as well as better clinical outcome reporting.

Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2[2] had reported the death of a patient on trial.

In A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer[3] authors: Lana E Kandalaft^*, Daniel J Powell and George Coukos from University of Pennsylvania recorded adverse events in pilot studies using a CART modified to recognize the folate receptor, so it appears any adverse event reporting system is at the discretion of the primary investigator.

Other protocol considerations suggested by the symposium attendants included:

Plan for translational clinical lab for routine blood analysis
Subject screening for pulmonary and cardiac events
Determine possibility of insertional mutagenesis
Informed consent
Analysis of non T and T-cell subsets, e.g. natural killer cells and CD*8 cells

3. Consideration for Design of Trials and Mitigating Toxicities

Early Toxic effects– Cytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).

Below is a video form Dr. Renier Brentjens, M.D., Ph.D. for Memorial Sloan Kettering concerning the finding he made that the adverse event from cytokine release syndrome may be a function of the tumor cell load, and if they treat the patient with CAR-T right after salvage chemotherapy the adverse events are alleviated..

Please see video below:

http link: https://www.youtube.com/watch?v=4Gg6elUMIVE

Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
Late Toxic Effects – long-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation

Below is a curation of various examples of the need for developing a Pharmacovigilence Framework for Engineered T-Cell Therapies

As shown above the first reported side effects from engineered T-cell or CAR-T therapies stemmed from the first human trial occuring at University of Pennsylvania, the developers of the first CAR-T therapy. The clinical investigators however anticipated the issue of a potential cytokine storm and had developed ideas in the pre-trial phase of how to ameliorate such toxicity using anti-cytokine antibodies. However, until the trial was underway they were unsure of which cytokines would be prominent in causing a cytokine storm effect from the CAR-T therapy. Fortunately, the investigators were able to save patient 1 (described here in other posts) using anti-IL1 and 10 antibodies.

Over the years, however, multiple trials had to be discontinued as shown below in the following posts:

What does this mean for Immunotherapy? FDA put a temporary hold on Juno’s JCAR015, Three Death of Celebral Edema in CAR-T Clinical Trial and Kite Pharma announced Phase II portion of its CAR-T ZUMA-1 trial

The NIH has put a crimp in the clinical trial work of Steven Rosenberg, Kite Pharma’s star collaborator at the National Cancer Institute. The feds slammed the brakes on the production of experimental drugs at two of its facilities–including cell therapies that Rosenberg works with–after an internal inspection found they weren’t in compliance with safety and quality regulations.

In this instance Kite was being cited for manufacturing issues, apparantly fungal contamination in their cell therapy manufacturing facility. However shortly after other CAR-T developers were having tragic deaths in their initial phase 1 safety studies.

Juno Halts Cancer Trial Using Gene-Altered Cells After 3 Deaths

By ANDREW POLLACKJULY 7, 2016

http://www.nytimes.com/2016/07/08/business/juno-halts-cancer-trial-using-gene-altered-cells-after-3-deaths.html?rref=collection%2Ftimestopic%2FFood%20and%20Drug%20Administration&action=click&contentCollection=timestopics&region=stream&module=stream_unit&version=latest&contentPlacement=2&pgtype=collection

Juno halts its immunotherapy trial for cancer after three patient deaths

By DAMIAN GARDE @damiangarde and MEGHANA KESHAVAN @megkesh

JULY 7, 2016

Juno halts its immunotherapy trial for cancer after three patient deaths

In Juno patient deaths, echoes seen of earlier failed company

By SHARON BEGLEY @sxbegle

JULY 8, 2016

https://www.statnews.com/2016/07/08/juno-echoes-of-dendreon/

After a deadly clinical trial, will immune therapies for cancer be a bust?

By DAMIAN GARDE @damiangarde

JULY 8, 2016

After a deadly clinical trial, will immune therapies for cancer be a bust?

This led to warnings by FDA and alteration of their trials as well as the use of their CART as a monotherapy

Hours after Juno CAR-T study deaths announced, Kite enrolls CAR-T PhII

Well That Was Quick! FDA Lets Juno Restart Trial With a New Combination Chemotherapuetic

Rachel Lerman at Seattle Times

FDA lets Juno restart cancer-treatment trial

Originally published July 12, 2016 at 4:07 pm Updated July 13, 2016 at 11:21 am

SOURCE: http://www.seattletimes.com/business/technology/fda-lets-juno-restart-cancer-treatment-trial/

Juno Therapeutics said Tuesday it will restart one of its most prominent clinical trials after the Food and Drug Administration lifted a hold that had been placed last week on the trial.

The FDA halted Juno’s “Rocket” clinical trial after the company reported thattwo patients undergoing treatment had died. Juno determined the deaths resulted from swelling in the brain caused by a new drug that had been added to the treatment.

he trial seeks to treat patients with relapsed acute lymphoblastic leukemia by using engineered T-cells to attack cancer cells.

Juno added the chemotherapy drug fludarabine to the treatment plan as part of an early step that gets the patient’s body ready for the T-cell injection. Previously, Juno was using only cyclophosphamide in that part of the treatment.

Under an agreement with the FDA, Juno will continue the trial without fludarabine, using only cyclophosphamide instead.

So What Did this all mean for the CAR-T world? Is it end for CAR-T Therapies?

NO!

This, as I have posted before is a matter of pharmacovigilence, the part of drug development and premarketing trials and postmarketing analysis that deals with adverse events and safety

see post of FDA guidelines for CAR-T therapy here

https://pharmaceuticalintelligence.com/2014/09/23/nih-considers-guidelines-for-car-t-therapy-report-from-recombinant-dna-advisory-committee/

The JUNO trial called for co-treatment with the drug fludarabine, and antimetabolite known, in some cases to promote a cytotoxic lysis syndrome and central nervous system complications. GRANTED these two side effects were deemed RARE however it appears that the addition of fludarabine pre CART therapy aggravated either the side effect of fludarabine pretreatment or a cytoxic lysis syndrome from CAR-T (an adverse event from CAR-T therapy as I had posted here INCLUDING REPORTS OF TWO DEATHS DURING THE MSK CAR-t TRIAL

https://pharmaceuticalintelligence.com/2015/09/14/steroids-inflammation-and-car-t-therapy/

Certainly with so many issues there would seem to be more rigorous work to either establish a pharmacovigilence framework or to develop alternative engineered T cells with a safer profile

However here we went again

New paper sheds fresh light on Tmunity’s high-profile CAR-T deaths
Jason Mast
Editor
The industry-wide effort to push CAR-T therapies — wildly effective in several blood cancers — into solid tumors took a hit last year when Tmunity, a biotech founded by CAR-T pioneer Carl June and backed by several blue-chip VCs, announced it shut down its lead program for prostate cancer after two patients died.

On a personal note this trial was announced in a Bio International meeting here in Philadelphia a few years ago in 2019

see Live Conference Coverage on this site

eProceedings for BIO 2019 International Convention, June 3-6, 2019 Philadelphia Convention Center; Philadelphia PA, Real Time Coverage by Stephen J. Williams, PhD @StephenJWillia2

and the indication was for prostate cancer, in particular hormone resistant castration resistant. Another one was planned for pancreatic cancer from the same group and the early indications were favorable.

From Onclive

Source: https://www.onclive.com/view/car-t-cell-therapy-trial-in-solid-tumors-halted-following-2-patient-deaths

Tmunity Therapeutics, a clinical-stage biotherapeutics company, has halted the development of its lead CAR T-cell product following the deaths of 2 patients who were enrolled to a trial investigating its use in solid tumors.¹

The patients reportedly died from immune effector cell-associated neurotoxicity syndrome (ICANS), which is a known adverse effect associated with CAR T-cell therapies.

“What we are discovering is that the cytokine profiles we see in solid tumors are completely different from hematologic cancers,” Oz Azam, co-founder of Tmunity said in an interview with Endpoints News. “We observed ICANS. And we had 2 patient deaths as a result of that. We navigated the first event and obviously saw the second event, and as a result of that we have shut down the version one of that program and pivoted quickly to our second generation.”

Previously, with first-generation CAR T-cell therapies in patients with blood cancers, investigators were presented with the challenge of overcoming cytokine release syndrome. Now ICANS, or macrophage activation, is proving to have deadly effects in the realm of solid tumors. Carl June, the other co-founder of Tmunity, noted that investigators will now need to dedicate their efforts to engineering around this, as had been done with tocilizumab (Actemra) in 2012.

The company is dedicated to the development of novel approaches that produce best-in-class control over T-cell activation and direction in the body.² The product examined in the trial was developed to utilize engineered patient cells to target prostate-specific membrane antigen; it was also designed to use a dominant TGFβ receptor to block an important checkpoint involved in cancer.

Twenty-four patients were recruited for the dose-escalating study and the company plans to release data from high-dose cohorts later in 2021.

“We are going to present all of this in a peer-reviewed publication because we want to share this with the field,” Azam said. “Because everything we’ve encountered, no matter what…people are going to encounter this when they get into the clinic, and I don’t think they’ve really understood yet because so many are preclinical companies that are not in the clinic with solid tumors. And the rubber meets the road when you get in the clinic, because the ultimate in vivo model is the human model.”

Azam added that the company plans to develop a new investigational new drug for version 2, which they hope will result in a safer product.

References

Carroll J. Exclusive: Carl June’s Tmunity encounters a lethal roadblock as 2 patient deaths derail lead trial, raise red flag forcing rethink of CAR-T for solid tumors. Endpoints News. June 2, 2021. Accessed June 3, 2021. https://bit.ly/3wPYWm0
Research and Development. Tmunity Therapeutics website. Accessed June 3, 2021. https://bit.ly/3fOH3OR

Forward to 2022

Reprogramming a new type of T cell to go after cancers with less side effects, longer impact

By Sophia SorensenApr 25, 2022 09:30am

A Sloan Kettering Institute research team thinks new, killer, innate-like T cells could make promising candidates to treat cancers that so far haven’t responded to immunotherapy treatments. (koto_feja)

Immunotherapy is one of the more appealing and effective kinds of cancer treatment when it works, but the relatively new approach is still fairly limited in the kinds of cancer it can be used for. Researchers at the Sloan Kettering Institute have discovered a new kind of immune cell and how it could be used to expand the reach of immunotherapy treatments to a much wider pool of patients.

The cells in question are called killer innate-like T cells, a threatening name for a potentially lifesaving innovation. Unlike normal killer T cells, killer innate-like T cells stay active much longer and can burrow further into potentially cancerous tissue to attack tumors. The research team first reported these cells in 2016, but it’s only recently that they were able to properly understand and identify them.

“We think these killer innate-like T cells could be targeted or genetically engineered for cancer therapy,” said the study’s lead author, Ming Li, Ph.D., in a press release. “They may be better at reaching and killing solid tumors than conventional T cells.”

Below is the referenced paper from Pubmed:

Evaluation of the safety and efficacy of humanized anti-CD19 chimeric antigen receptor T-cell therapy in older patients with relapsed/refractory diffuse large B-cell lymphoma based on the comprehensive geriatric assessment system

Huan Zhang¹, Man Liu², Qing Li¹, Cuicui Lyu¹, Yan-Yu Jiang¹, Juan-Xia Meng¹, Jing-Yi Li¹, Qi Deng¹

Affiliations

PMID: 34587859
DOI: 10.1080/10428194.2021.1986216

Abstract

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has led to unprecedented results to date in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), yet its clinical application in elderly patients with R/R DLBCL remains somewhat limited. In this study, a total of 31 R/R DLBCL patients older than 65 years of age were enrolled and received humanized anti-CD19 CAR T-cell therapy. Patients were stratified into a fit, unfit, or frail group according to the comprehensive geriatric assessment (CGA). The fit group had a higher objective response (OR) rate (ORR) and complete response (CR) rate than that of the unfit/frail group, but there was no difference in the part response (PR) rate between the groups. The unfit/frail group was more likely to experience AEs than the fit group. The peak proportion of anti-CD19 CAR T-cells in the fit group was significantly higher than that of the unfit/frail group. The CGA can be used to effectively predict the treatment response, adverse events, and long-term survival.

Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30–40% of cases, with the median age of onset being older than 65 years [1]. Although the five-year survival rate for patients with DLBCL has risen to more than 60% with the application of standardized treatments and hematopoietic stem cell transplantation, nearly half of patients progress to relapsed/refractory (R/R) DLBCL. Patients with R/R DLBCL, especially elderly individuals, have a poor prognosis [2,3], so new treatments are needed to prolong survival and improve the prognosis of this population.

As a revolutionary immunotherapy therapy, anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has achieved unprecedented results in hematological tumors [4]. As CD19 is expressed on the surface of most B-cell malignant tumors but not on pluripotent bone marrow stem cells, CD19 has been used as a target for B-cell malignancies, including B-cell acute lymphoblastic leukemia, NHL, multiple myeloma, and chronic lymphocytic leukemia [5]. Despite the wide application and high efficacy of anti-CD19 CAR T-cell therapy, reports of adverse events (AEs) such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxic syndrome (ICANS) have influenced its use [6]. Especially in elderly patients, AEs associated with anti-CD19 CAR T-cell therapy might be more obvious.

Although anti-CD19 CAR T-cell therapy has been reported in the treatment of NHL, including R/R DLBCL, few studies to date have assessed the safety of anti-CD19 CAR T-cell therapy in elderly R/R DLBCL patients, and its clinical application in the elderly R/R DLBCL population is limited. In ZUMA-1 [7] to R/R DLBCL patients who received CAR T-cell therapy, the CR rate in patients ≥65 years was higher than that of in patients <65 years (75% vs. 53%). Lin et al. [8] reported 49 R/R DLBCL patients (24 patients >65 years, 25 patients <65 years) who received CAR T-cell therapy with a median follow-up of 179 days. The CR rate at 100 days was 51%, while the 6-month progression-free survival (PFS) and overall survival (OS) were 48% and 71%, respectively. Neither of the two studies carried out a comprehensive geriatric assessment (CGA) of fit, unfit, and frail groups of R/R DLBCL patients over 65 years of age and further analyzed the differences in efficacy and side effects in the three groups. The CGA is an effective system designed to evaluate the prognosis and improve the survival of elderly patients with cancer. The CGA system includes age, activities of daily living (ADL), instrumental ADL (IADL), and the Cumulative Illness Rating Score for Geriatrics (CIRS-G) [9].

In this study, elderly R/R DLBCL patients were grouped according to their CGA results (fit vs. unfit/frail) before receiving humanized anti-CD19 CAR T-cell therapy. We then analyzed the efficacy and AEs of anti-CD19 CAR T-cell therapy and compared findings between these groups.

Well it appears that the discriminator was only fitness going into the trial a bit odd that the whole field appears to be lacking in development of Safety Biomarkers.

However Genentech (subsidiary of Roche) may now be using some data to develop therapies which may combat resistance to CART therapies which may provide at least, for now, a toxicokinetic approach to reducing AEs by lowering the amount of CARTs needed to be administered.

Source: https://www.fiercebiotech.com/research/genentech-uncovers-how-cancer-cells-resist-t-cell-attack-potential-boon-immunotherapy

Roche’s Genentech is exploring inhibiting ESCRT as an anticancer strategy, said Ira Mellman, Ph.D., Genentech’s vice president of cancer immunology. (Roche)

Cancer cells deploy various tactics to avoid being targeted and killed by the immune system. A research team led by Roche’s Genentech has now identified one such method that cancer cells use to resist T-cell assault by repairing damage.

To destroy their targets, cancer-killing T cells known as cytotoxic T lymphocytes (CTLs) secrete the toxin perforin to form little pores in the target cells’ surface. Another type of toxin called granzymes are delivered directly into the cells through those portals to induce cell death.

By using high-res imaging in live cells, the Genentech-led team found that the membrane damage caused by perforin could trigger a repair response. The tumor cells could recruit endosomal sorting complexes required for transport (ESCRT) proteins to remove the lesions, thereby preventing granzymes from entering, the team showed in a new study published in Science.

The following is the Science paper

Source: https://www.science.org/doi/10.1126/science.abl3855

Membrane repair in target cell defenses

Killer T cells destroy virus-infected and cancer cells by secreting two protein toxins that act as a powerful one-two punch. Pore-forming toxins, perforins, form holes in the plasma membrane of the target cell. Cytotoxic proteins released by T cells then pass through these portals, inducing target cell death. Ritter et al. combined high-resolution imaging data with functional analysis to demonstrate that tumor-derived cells fight back (see the Perspective by Andrews). Protein complexes of the ESCRT family were able to repair perforin holes in target cells, thereby delaying or preventing T cell–induced killing. ESCRT-mediated membrane repair may thus provide a mechanism of resistance to immune attack. —SMH

Abstract

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.

Cytotoxic lymphocytes, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, are responsible for identifying and destroying virus-infected or tumorigenic cells. To kill their targets, CTLs and NK cells secrete a pore-forming toxin called perforin through which apoptosis-inducing serine proteases (granzymes) are delivered directly into the cytosol. Successful killing of target cells often requires multiple hits from single or multiple T cells (1). This has led to the idea that cytotoxicity is additive, often requiring multiple rounds of sublethal lytic granule secretion events before a sufficient threshold of cytosolic granzyme activity is reached to initiate apoptosis in the target (2).

Loss of plasma membrane integrity induced by cytolytic proteins or mechanical damage leads to a membrane repair response. Damage results in an influx of extracellular Ca²⁺, which has been proposed to lead to the removal of the membrane lesion by endocytosis, resealing of the lesions by lysosomal secretion, or budding into extracellular vesicles (3). Perforin pore formation was initially reported to enhance endocytosis of perforin (4), but subsequent work has challenged this claim (5). Endosomal sorting complexes required for transport (ESCRT) proteins can repair small wounds and pores in the plasma membrane caused by bacterial pore-forming toxins, mechanical wounding, and laser ablation (6, 7). ESCRT proteins are transiently recruited to sites of membrane damage in a Ca²⁺-dependent fashion, where they assemble budding structures that shed to eliminate the wound and restore plasma membrane integrity. ESCRT-dependent membrane repair has been implicated in the resealing of endogenous pore-mediated plasma membrane damage during necroptosis (8) and pyroptosis (9).

Localization of target-derived ESCRT proteins to the cytolytic synapse

To investigate whether ESCRT-mediated membrane repair might be involved in the removal of perforin pores during T cell killing, we first determined whether ESCRT proteins in cancer-derived cells were recruited to sites of CTL engagement after perforin secretion. We used CTLs from OT-I mice that express a high-affinity T cell receptor (TCR) that recognizes the ovalbumin peptide SIINFEKL (OVA_257-264) bound to the major histocompatibility complex (MHC) allele H-2Kb (10). We performed live-cell microscopy of OT-I CTLs engaging SIINFEKL-pulsed target cells that express enhanced green fluorescent protein (EGFP)–tagged versions of Tsg101 or Chmp4b, two ESCRT proteins implicated in membrane repair (6). To correlate recruitment of ESCRT proteins with perforin exposure in time, we monitored CTL-target interaction in media with a high concentration of propidium iodide (PI), a cell-impermeable fluorogenic dye that can rapidly diffuse through perforin pores to bind and illuminate nucleic acids in the cytosol and nucleus of the target (5). EGFP-tagged ESCRT proteins were consistently recruited to the site of CTL engagement within 30 to 60 s after PI influx (Fig. 1, A and B). EGFP-Tsg101 and EGFP-Chmp4b in target cells accumulated at the cytolytic synapse after PI influx in 25 of 27 (92.6%) and 31 of 33 (93.9%) of conjugates monitored, respectively, compared with a cytosolic EGFP control, which was not recruited (Fig. 1C and movies S1 to S3). Notably, ESCRT-laden material, presumably membrane fragments, frequently detached from the target cell and adhered to the surface of the CTL (Fig. 1, D and E, and movie S2). We observed this phenomenon in ~60% of conjugates imaged in which targets expressed EGFP-Tsg101 or EGFP-Chmp4b (17 of 27 and 20 of 33 conjugates, respectively; Fig. 1D). Shedding of ESCRT-positive membrane from the cell after repair occurs after laser-induced plasma membrane wounding (6, 7). Plasma membrane fragments shed from the target cell into the synaptic cleft likely contain ligands for CTL-resident receptors. Target cell death would separate the CTL and target, revealing target-derived material on the CTL surface.

FIG. 1. Fluorescently tagged ESCRT proteins in targets localize to site of CTL killing after perforin secretion.

(A) Live-cell spinning disk confocal imaging of a fluorescently labeled OT-I CTL (magenta) engaging an MC38 cancer cell expressing EGFP-Tsg101 (green) in media containing 100 μM PI (red). Yellow arrowheads highlight ESCRT recruitment. T-0:00 is the first frame of PI influx into the target cell (time in minutes:seconds). Scale bar, 10 μm. (B) Graph of EGFP-Tsg101 and PI fluorescence intensity at the IS within the target over time, from example in (A). AU, arbitrary units. (C and D) Quantification of CTL-target conjugates exhibiting accumulation of EGFP at the synapse after PI influx (C) or detectable EGFP-labeled material associated with CTL after target interaction (D) (EGFP condition: N = 22 conjugates in seven independent experiments; EGFP-Tsg101 condition: N = 27 conjugates in nine independent experiments; EGFP-Chmp4b condition: N = 33 conjugates in 24 independent experiments). (E) Live-cell spinning disk confocal imaging of OT-I CTL (magenta) killing MC38 expressing EGFP-Chmp4b (green), demonstrating the presence of target-derived EGFP-Chmp4b material (yellow arrowheads) associated with CTL membrane after a productive target encounter. T-0:00 is the first frame of PI influx into the target cell. Scale bar, 10 μm.

3D cryo-SIM and FIB-SEM imaging of CTLs caught in the act of killing target cells

Although live-cell imaging indicated that ESCRT complexes were rapidly recruited at sites of T cell–target cell contact, light microscopy alone is of insufficient resolution to establish that this event occurred at the immunological synapse (IS). We thus sought to capture a comprehensive view of the IS in the moments immediately after secretion of lytic granules. We used cryo–fluorescence imaging followed by correlative focused ion beam–scanning electron microscopy (FIB-SEM), which can achieve isotropic three-dimensional (3D) imaging of whole cells at 8-nm resolution or better (11–13). To capture the immediate response of target cells after perforin exposure, we developed a strategy whereby cryo-fixed CTL-target conjugates were selected shortly after perforation, indicated by the presence of a PI gradient in the target (fig. S1A). In live-cell imaging experiments, PI fluorescence across the nucleus of SIINFEKL-pulsed ID8 target cells began as a gradient and became homogeneous 158 ± 64 s, on average, after initial PI influx (N = 31 conjugates; fig. S1, B and C, and movie S4). Thus, fixed CTL-target conjugates that exhibited a gradient of PI across the nucleus would have been captured within ~3 min of perforin exposure.

Coverslips of CTL-target conjugates underwent high-pressure freezing and were subsequently imaged with wide-field cryogenic fluorescence microscopy followed by 3D cryo–structured illumination microscopy (3D cryo-SIM) performed in a customized optical cryostat (14). We selected candidate conjugates for FIB-SEM imaging on the basis of whether a gradient of PI fluorescence was observed across the nucleus of the target emanating from an attached CTL (movie S5). FIB-SEM imaging of the CTL-target conjugate at 8-nm isotropic voxels resulted in a stack of >10,000 individual electron microscopy (EM) images. The image stack was then annotated using a human-assisted machine learning–computer vision platform to segment the plasma membranes of each cell along with cell nuclei and various organelles (https://ariadne.ai/).

We captured four isotropic 3D 8-nm-resolution EM datasets of CTLs killing cancer cells moments after the secretion of lytic granule contents (Fig. 2A and movie S6). Semiautomated segmentation of the cell membranes, nuclei, lytic granules, Golgi apparatus, mitochondria, and centrosomes of the T cells allow for easier visualization and analysis of the 3D EM data. All FIB-SEM datasets and segmentations can be explored online at https://openorganelle.janelia.org (see links in the supplementary materials). Reconstructed views of the segmented data clearly demonstrate the polarization of the centrosome, Golgi apparatus, and lytic granules to the IS—all of which are hallmarks of CTL killing [Fig. 2A, i to iii, and movie S6, time stamp (TS) 1:33] (15, 16). On the target cell side, we noted cytoplasmic alterations consistent with cell damage including enhanced electron density of mitochondria adjacent to the IS (fig. S2A). Close visual scanning of the postsynaptic target cell membrane in the raw EM data failed to reveal obvious perforin pores, which have diameters (16 to 22 nm) close to the limit of resolution for this technique (17).

FIG. 2. Eight-nm-resolution 3D FIB-SEM imaging of whole CTL-target conjugate.

(A) 3D rendering of segmented plasma membrane predictions derived from isotropic 8-nm-resolution FIB-SEM imaging of a high-pressure frozen OT-I CTL (red) captured moments after secretion of lytic granules toward a peptide-pulsed ID8 ovarian cancer cell (blue). (i) Side-on sliced view corresponding to the gray horizontal line within the inset box in (A). Seen here are 3D renderings of the segmented plasma membrane of the cancer cell (blue) as well as the CTL plasma membrane (red), centrosome (gold), Golgi apparatus (cyan), lytic granules (purple), mitochondria (green), and nucleus (gray). (ii and iii) A zoomed-in view from the dashed white box in (i) shows the details of the IS (ii) and a single corresponding FIB-SEM slice docked onto the segmented data (iii). (B) Single top-down FIB-SEM slice showing overlaid target cell (blue) and CTL (red) segmentation. (i) Zoomed-in view from dashed white box in (B) details the intercellular material (IM) (gray) between the CTL and target at the IS. (C) Zoomed-in image of a 3D rendering of the surface of the target cell plasma membrane (white) opposite the intercellular material (IM) at the IS. Yellow arrowheads mark plasma membrane buds protruding into the synaptic cleft. (i and ii) Accompanying images demonstrate the orientation of the view in (C) with the rendering of the CTL (red) present (i) and removed (ii), and the dashed yellow box in (ii) indicates the area of detail shown in (C).

The segmentation of the two cells illustrates the detailed topography of the plasma membrane of the CTL and target at the IS (fig. S2B). The raw EM and segmentation data reveal a dense accumulation of particles, vesicles, and multilamellar membranous materials, which crowd the synaptic cleft between the CTL and the target (Fig. 2B and movie S6, TS 0:40 to 0:50). The source of this intercellular material (IM) was likely in part the lytic granules because close inspection revealed similar particles and dense vesicles located within as-yet-unreleased granules (fig. S2C). To determine whether some of the membranous material within the intercellular space might also have been derived from the target cell, we examined the surface topology of the postsynaptic target cell. We noted multiple tubular and bud-like protrusions of the target cell membrane that extended into the synaptic space; thus, at least some of the membrane structures observed were still in continuity with the target cell (Fig. 2C and movie S6, TS 0:58 to 1:11). ESCRT proteins have been shown to generate budding structures in the context of plasma membrane repair (6), which led us to next assess where target-derived ESCRT proteins are distributed in the context of the postsecretion IS.

To map the localization of target-derived ESCRT proteins onto a high-resolution landscape of the IS, we captured three FIB-SEM datasets that have associated 3D cryo-SIM fluorescence data for mEmerald-Chmp4b localization (Fig. 3A, fig. S3, and movie S7). This correlative light and electron microscopy (CLEM) revealed that mEmerald-Chmp4b expressed in the target cell was specifically recruited to the target plasma membrane opposite the secreted IM (Fig. 3, B and C). The topography of the plasma membrane at the site of ESCRT recruitment was markedly convoluted, exhibiting many bud-like projections (movie S7, TS 0:37 to 0:40). mEmerald-Chmp4b fluorescence also overlapped with some vesicular structures in the intercellular synaptic space (Fig. 3C). Together, the live-cell imaging and the 3D cryo-SIM and FIB-SEM CLEM demonstrate the localization of ESCRT proteins at the synapse that was the definitive site of CTL killing and was thus spatially and temporally correlated to perforin secretion. These data implicate the ESCRT complex in the repair of perforin pores.

FIG. 3. Correlative 3D cryo-SIM and FIB-SEM reveal localization of target-derived ESCRT within the cytolytic IS.

(A) Three example datasets showing correlative 3D cryo-SIM and FIB-SEM imaging of OT-I CTLs (red) captured moments after secretion of lytic granules toward peptide-pulsed ID8 cancer cells (blue) expressing mEmerald-Chmp4b (green fluorescence). (B and C) Single FIB-SEM slices corresponding to the orange boxes in (A), overlaid with CTL and cancer cell segmentation (B) or correlative cryo-SIM fluorescence of mEmerald-Chmp4b derived from the target cell (C).

Function of ESCRT proteins in repair of perforin pores

We next investigated whether ESCRT inhibition could enhance the susceptibility of target cells to CTL-mediated killing. Prolonged inactivation of the ESCRT pathway is itself cytotoxic (9). We thus developed strategies to ablate ESCRT function that would allow us a window of time to assess CTL killing (fig. S4). We used two approaches to block ESCRT function: CRISPR knockout of the Chmp4b gene or overexpression of VPS4a^E228Q (E228Q, Glu²²⁸ → Gln), a dominant-negative kinase allele that impairs ESCRT function (fig. S4, A to C) (10). We took care to complete our assessment of target killing well in advance of spontaneous target cell death (fig. S4D).

We tested the capacity of OT-I CTLs to kill targets presenting one of four previously characterized peptides that demonstrate a range of potencies at stimulating the OT-I TCR: SIINFEKL (N4), the cognate peptide, and three separate variants (in order of highest to lowest affinity), SIITFEKL (T4), SIIQFEHL (Q4H7), and SIIGFEKL (G4) (18, 19). Target cells were pulsed with peptide, washed, transferred to 96-well plates, and allowed to adhere before the addition of OT-I CTLs. Killing was assessed by monitoring the uptake of a fluorogenic caspase 3/7 indicator (Fig. 4, A to D, and fig. S5A). Killing was significantly more efficient in ESCRT-inhibited target cells for both CRISPR depletion of Chmp4b (Fig. 4, A to C) and expression of the dominant-negative VPS4a^E228Q (Fig. 4D). The difference in killing between the ESCRT-inhibited and control cells was greater when the lower-potency T4, Q4H7, and G4 peptides were used. Nevertheless, ESCRT inhibition moderately improved killing efficiency even in the case of the high-potency SIINFEKL peptide. ESCRT inhibition had no effect on MHC class I expression on the surface of target cells (fig. S5B). Thus, ESCRT inhibition could sensitize target cells to perforin- and granzyme-mediated killing, especially at physiologically relevant TCR-peptide MHC affinities.

FIG. 4. ESCRT inhibition enhances susceptibility of cancer cells to CTL killing and recombinant lytic proteins.

(A) Representative time-lapse data of killing of peptide-pulsed Chmp4b knockout (KO) or control B16-F10 cells by OT-I CTLs. Affinity of the pulsed peptide to OT-I TCR decreases from left to right. Error bars indicate SDs. (B) Images extracted from T4 medium-affinity peptide condition show software-detected caspase 3/7+ events in control and Chmp4b KO conditions. (C and D) Data representing the 4-hour time point of assays measuring OT-I T cells killing either Chmp4b KO (C) or VPS4 dominant-negative (D) target cells with matched controls. Error bars indicate SDs of data. Data are representative of at least three independent experimental replicates. pMHC, peptide-MHC; HA, hemagglutinin. (E and F) Determination of sublytic dose of Prf. B16-F10 cells expressing VPS4a (WT or E228Q) were exposed to increasing concentrations of Prf. Cell viability was determined by morphological gating (E). FSC, forward scatter; SSC, side scatter. (G and H) B16-F10 cells expressing VPS4a (WT or E228Q) were exposed to a sublytic dose of Prf in combination with increasing concentrations of recombinant GZMB (rGZMB). Cell death was determined by Annexin V–allophycocyanin (APC) staining (G). Controls include a condition with no perforin and 5000 ng/ml rGZMB and sublytic perforin with no rGZMB. Graphs in (F) and (H) represent the means of three experiments, and error bars indicate SDs. Statistical significance was determined by multiple unpaired t tests with alpha = 0.05. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

We next directly tested the effects of ESCRT inhibition when target cells were exposed to both recombinant perforin (Prf) and granzyme B (GZMB), the most potently proapoptotic granzyme in humans and mice (20). Prf alone at high concentrations can lyse cells (4), so we first determined a sublytic Prf concentration that would temporarily permeabilize the plasma membrane but permit the cells to recover. B16-F10 cells expressing either VPS4a^WT (WT, wild-type) or VPS4a^E228Q were exposed to a range of Prf concentrations in the presence of PI, and cell viability and PI uptake were assessed using flow cytometry. Cells that expressed dominant-negative VPS4a^E228Q were more sensitive to Prf alone than ESCRT-competent cells (Fig. 4, E and F). At 160 ng/ml Prf, there was no significant difference in cell viability for either condition. Cells in the live gate that were PI+ had been permeabilized by Prf but recovered. Although the percentage of PI+ live cells was similar under both sets of conditions, the mean fluorescence intensity of PI was higher in live ESCRT-inhibited cells (fig. S6). A delay in plasma membrane resealing could account for this difference.

We reasoned that delaying perforin pore repair might also enhance GZMB uptake into the target. ESCRT-inhibited cells were more sensitive to combined perforin-GZMB when cell death was measured by Annexin V staining (Fig. 4, G and H). Similar results were observed when these experiments were repeated with a murine lymphoma cancer cell line (fig. S7). The observation that ESCRT-inhibited target cells are more sensitive to both CTL-secreted and Prf-GZMB supports the hypothesis that the ESCRT pathway contributes to membrane repair after Prf exposure.

Escaping cell death is one of the hallmarks of cancer. Our findings suggest that ESCRT-mediated membrane repair of perforin pores may restrict accessibility of the target cytosol to CTL-secreted granzyme, thus promoting survival of cancer-derived cells under cytolytic attack. Although other factors may contribute to setting the threshold for target susceptibility to killing, the role of active repair of perforin pores must now be considered as a clear contributing factor.

Acknowledgments

We thank members of the Mellman laboratory for advice, discussion, and reagents; B. Haley for assistance with plasmid construct design; the Genentech FACS Core Facility for technical assistance; S. Van Engelenburg of Denver University for invaluable discussions and guidance; A. Wanner, S. Spaar, and the Ariande AI AG (https://ariadne.ai/) for assistance with FIB-SEM segmentation, CLEM coregistration, data presentation, and rendering; D. Bennett of the Janelia Research Campus for assisting with data upload to https://openorganelle.janelia.org; and the Genentech Postdoctoral Program for support.

Funding: A.T.R. and I.M. are funded by Genentech/Roche. C.S.X., G.S., A.W., D.A., N.I., and H.F.H. are funded by the Howard Hughes Medical Institute (HHMI).

Please look for a Followup Post concerning “Developing a Pharmacovigilence Framework for Engineered T-Cell Therapies”

References

Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

Other posts on this site on Immunotherapy and Cancer include

Report on Cancer Immunotherapy Market & Clinical Pipeline Insight

New Immunotherapy Could Fight a Range of Cancers

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Molecular Profiling in Cancer Immunotherapy: Debraj GuhaThakurta, PhD

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

$20 million Novartis deal with ‘University of Pennsylvania’ to develop Ultra-Personalized Cancer Immunotherapy

Upcoming Meetings on Cancer Immunogenetics

Tang Prize for 2014: Immunity and Cancer

ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors @DM Anderson Cancer Center

Juno’s approach eradicated cancer cells in 10 of 12 leukemia patients, indicating potential to transform the standard of care in oncology

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Calcium-Channel Blocker, Calcium as Neurotransmitter Sensor and Calcium Release-related Contractile Dysfunction (Ryanopathy)

Posted in Calcium Signaling, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, tagged Aviva Lev-Ari, Bernard Katz, Calcium-Channel Blocker, gene therapy, Heart Failure, Larry H. Bernstein, Michael J Berridge, Smooth muscle tissue, Stanford University, Vascular smooth muscle on September 16, 2013| Leave a Comment »

Calcium-Channel Blocker, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

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

Author and Curator: Larry H Bernstein, MD, FCAP

and Article Curator: Aviva Lev-Ari, PhD, RN

Article IX Calcium-Channel Blocker, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Image created by Adina Hazan 06/30/2021

Introduction

Author: Larry H Bernstein, MD, FACC

This Chapter is one of a series of articles on calcium activation, in this case, in the signaling of smooth muscle cells by the interacting neural innervation. The process occurs by calcium triggering neurotransmitter release by initiating synaptic vesicle fusion. The mechanism by which this occurs is addressed in detail, and involves the interaction of soluble N-acetylmaleimide-sensitive factor (SNARE) and SM proteins, and in addition, the discovery of a calcium-dependsent Syt1 (C) domain of protein- kinase C isoenzyme, which binds to phospholipids.

The 2013 Lasker Prize was awarded to Richard Schell (Genentech) and Thomas Sudolf (Stanford University) for their discoveries concerning the molecular machinery and regulatory mechanism that underlie the rapid release of neurotransmitters, a process that underlies all of the brain’s activities. They identified and isolated many of this reaction’s key elements, unraveled central aspects of its fundamental mechanism, and deciphered how cells govern it with extreme precision. These advances have provided a molecular framework for understanding some of the most devastating disorders that afflict humans as well as normal functions such as learning and memory, explaining unresolved hypotheses derived from the earlier work in the 1950sof the late Bernard Katz. We also see that the work clarifies the debates initiated by the Nobelist Santiago Ramon y Cajal (1891) concerning the development of neural networks. A biological relay system achieves these feats. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers—neurotransmitters.

In the 1950s, the late Bernard Katz figured out that cells eject neurotransmitters in fixed amounts. Balloon-like containers—vesicles—each hold set quantities of the chemicals. Calcium incites these lipid-bound sacs to fuse with the membrane that encases the cell, and their contents spill out. The picture that emerges from the later work is that synaptic vesicle exocytosis operates by a general mechanism of membrane fusion that revealed itself to be a model for all membrane fusion, but that is uniquely regulated by a calcium-sensor protein called synaptotagmin. The general membrane-fusion mechanism thus identified is mediated by SNARE- (for soluble NSF-receptors) and SM-proteins (for Sec1/Munc18-like proteins), largely discovered at the synapse, with synaptotagmin acting together with a molecular assistant called complexin as a clamp and activator of the membrane fusion mediated by the SNARE- and SM-proteins. Strikingly, the biochemical properties of synaptotagmin were found to precisely correspond to the extraordinary calcium-triggering properties of release, and to account for a regulatory pathway that also applies to other types of calcium-triggered fusion, for example fusion observed in hormone secretion and fertilization. At the synapse, finally, these interdependent machines — the fusion apparatus and its synaptotagmin-dependent control mechanism — are embedded in a proteinaceous active zone that links them to calcium channels, and regulates the docking and priming of synaptic vesicles for subsequent calcium-triggered fusion. Thus, work on neurotransmitter release revealed a hierarchy of molecular machines that mediate the fusion of synaptic vesicles, the calcium-control of this fusion, and the embedding of calcium-controlled fusion in the context of the presynaptic terminal at the synapse.

This portion of the discussion deals with the interaction of the neuronal endings interwoven into smooth muscle. The calcium triggering of smooth muscle contractions is similar with respect to airways and arteries, urinary bladder, uterine contraction, and gastrointestinl tract.

The basic mechanism that underlie this MOTIF taken as variations of that described above are well described by Michael J. Berridge in ‘Smooth muscle cell calcium activation mechanisms’. (J Physiol. 2008 Nov 1;586(Pt 21):5047-61.
http://dx.doi.org/10.1113/jphysiol.2008.160440. Epub 2008 Sep 11.)

This is illustrated in his graphical examples.

http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Figure 1. The three main mechanisms responsible for generating the Ca2+ transients that trigger smooth muscle cell (SMC) contraction. From: Smooth muscle cell calcium activation mechanisms.

A, receptor-operated channels (ROCs) or a membrane oscillator induces the membrane depolarization (ΔV) that triggers Ca2+ entry and contraction.

B, a cytosolic Ca2+ oscillator induces the Ca2+ signal that drives contraction.

C, a cytosolic Ca2+ oscillator in interstitial cells of Cajal (ICCs) or atypical SMCs induces the membrane depolarization that spreads through the gap junctions to activate neighbouring SMCs. Reproduced from Berridge (2008), with permission.

Michael J Berridge. J Physiol. 2008 November 1;586(Pt 21):5047-5061. http://www.ncbi.nlm.nih.gov/pmc/articles/instance/2652144/bin/tjp0586-5047-f1.jpg

Figure 5. Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave. From: Smooth muscle cell calcium activation mechanisms.

The oscillator is induced/modulated by neurotransmitters such as acetylcholine (ACh), 5-hydroxytryptamine (5-HT), noradrenaline (NA) and endothelin-1 (ET-1), which act through inositol 1,4,5-trisphosphate (InsP3) that initiates the oscillatory mechanism. The sequence of steps 1–9 is described in the text. Reproduced from Berridge (2008), with permission.
Michael J Berridge. J Physiol. 2008 November 1;586(Pt 21):5047-5061. http://www.ncbi.nlm.nih.gov/pmc/articles/instance/2652144/bin/tjp0586-5047-f5.jpg

Figure 7. The cytosolic Ca2+ oscillator responsible for pacemaker activity in interstitial cells of Cajal releases periodic pulses of Ca2+ that form a Ca2+ wave. From: Smooth muscle cell calcium activation mechanisms.

tjp0586-5047-f7 The cytosolic Ca2+ oscillator responsible for pacemaker activity in interstitial cells of Cajal releases periodic pulses of Ca2+ that form a Ca2+ wave.

The increase in Ca2+ activates Cl− channels (CLCA) to give the spontaneous transient inward currents (STICs) that sum to form the spontaneous transient depolarizations (STD) resulting in the slow waves of membrane depolarization (see inset). Current flow through gap junctions allows these waves to spread into neighbouring smooth muscle cells to activate contraction. See text for a description of the oscillator that drives this activation process. Reproduced from Berridge (2008), with permission.
Michael J Berridge. J Physiol. 2008 November 1;586(Pt 21):5047-5061. http://www.ncbi.nlm.nih.gov/pmc/articles/instance/2652144/bin/tjp0586-5047-f7.gif

This article is the Part IX in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells.

The Series consists of the following articles:

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

Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

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

http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

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

http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

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

http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

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

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

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

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/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmiasand 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

http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

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

http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

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

http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: 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/

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This article has FIVE Sections:

Section One

Innovations in Combination Drug Therapy: Calcium-Channel Blocker – Amlodipine (Norvasc) in single-pill combinations (SPCs) of drugs

Section Two

Calcium-Channel Blockers: Drug Class and Indications

Section Three

Brand and Generic Calcium Channel Blocking Agents

Section Four

Dysfunction of the Calcium Release Mechanism

Section Five

The Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Section One

Innovations in Combination Drug Therapy:

Calcium-Channel Blocker, Amlodipine (Norvasc) in Single-Pill Combinations (SPCs) of Drugs

Latest development on Cardiovascular Pharmacotherapy relates to the development of a Duo Combination Therapy to include a leading Calcium-Channel Blocker, Amlodipine (Norvasc), as one of the two drug classes in one pill:The research investigated the therapeutic efficacy achieved via a comparison of a two single-pill combinations (SPCs) of drugs:

telmisartan/amlodipine (T/A) [ARB/CCB]

and

telmisartan/hydrochlorothiazide (T/H) [ARB/Diuterics]

Drug classes:

ARB – telmisartan
CCB – amlodipine
Diuretics – hydrochlorothiazide

A review of the benefits of early treatment initiation with single-pill combinations of telmisartan with amlodipine or hydrochlorothiazide

Authors: Segura J, Ruilope LM

Published Date September 2013 Volume 2013:9 Pages 521 – 528

http://www.dovepress.com/articles.php?article_id=14373

DOI: http://dx.doi.org/10.2147/VHRM.S48291


Published:	16 September 2013, Dovepress Journal: Vascular Health and Risk Management

Julian Segura, Luis Miguel Ruilope

Department of Nephrology, Hospital 12 de Octubre, Madrid, Spain

Abstract:

This review discusses the rationale for earlier use of single-pill combinations (SPCs) of antihypertensive drugs, with a focus on telmisartan/amlodipine (T/A) and telmisartan/hydrochlorothiazide (T/H) SPCs.

Compared with the respective monotherapies, the once-daily T/A and T/H SPCs have been shown to result in significantly higher blood pressure (BP) reductions, BP goal rates, and response rates in patients at all stages of hypertension.

As expected, BP reductions are highest with the highest dose (T80/A10 and T80/H25) SPCs. Subgroup analyses of the telmisartan trials have reported the efficacy of both SPCs to be consistent, regardless of the patients’ age, race, and coexisting diabetes, obesity, or renal impairment.

In patients with mild-to-moderate hypertension, the T/A combination provides superior 24-hour BP-lowering efficacy compared with either treatment administered as monotherapy.

Similarly, the T/H SPC treatment provides superior 24-hour BP-lowering efficacy, especially in the last 6 hours relative to other renin–angiotensin system inhibitor-based SPCs.

The T/A SPC is associated with a lower incidence of edema than amlodipine monotherapy, and

The T/H SPC with a lower incidence of hypokalemia than hydrochlorothiazide monotherapy

Existing evidence supports the use of the T/A SPC for the treatment of hypertensive patients with prediabetes, diabetes, or metabolic syndrome, due to the metabolic neutrality of both component drugs, and the use of the T/H SPC for those patients with edema or in need of volume reduction.

Keywords: angiotensin receptor blockers, or ARBs, calcium-channel blocker, or CCBs, essential hypertension, diuretic, , renin-angiotensin system inhibitor, or ACEI

http://www.dovepress.com/articles.php?article_id=14373
We reported on 5/29/2012

Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

Triple Combination Therapy: ARB and Calcium Channel Blocker and Diuretics

In July 2010, a triple combination drug for hypertension was approved by the US Food and Drug Administration. Tribenzor contains olmesartan medoxomil, amlodipine and hydrochlorothiazide, according to Monthly Prescribing Reference.

http://pharmaceuticalintelligence.com/2012/05/29/445/

TRIBENZOR is a Daiichi Sankyo’s product- ARB and Calcium Channel Blocker and Diuretic

How TRIBENZOR work

Tribenzor contains olmesartan medoxomil, amlodipine and hydrochlorothiazide. High blood pressure makes the heart work harder to pump blood through the body and causes damage to blood vessels. TRIBENZOR can help your blood vessels relax and reduce the amount of fluid in your blood. This can make your blood pressure lower. Medicines that lower blood pressure may lower your chance of having a stroke or a heart attack.

Some people may need more than 1—or even more than 2—medicines to help control their blood pressure. TRIBENZOR combines 3 effective medicines in 1 convenient pill. Read the following chart to learn how each medicine works in its own way to help lower blood pressure.

TRIBENZOR: 3 effective medicines in 1 pill

The medicine in TRIBENZOR	How it works	What it does
Angiotensin II receptor blocker	Blocks a natural chemical in your body that causes blood vessels to narrow.	Lowers Yours blood pressure
Calcium channel blocker	Blocks the narrowing effect of calcium on your blood vessels. This helps your blood vessels relax.
Diuretic (water pill)	Helps your kidneys flush extra fluid and salt from your body. This lowers the amount of fluid in your blood.

http://www.tribenzor.com/how_works.html

Effectively lower blood pressure. People taking the 3 medicines in TRIBENZOR had greater reductions in blood pressure than did people taking any 2 of the medicines combined

Start to work quickly. People taking TRIBENZOR saw results in as little as 2 weeks

AZOR is a Daiichi Sankyo’s product- ARB and Calcium Channel Blocker

How AZOR work

AZOR relaxes and widens blood vessels to help lower blood pressure.

You may have already tried another blood pressure medicine that works a certain way to lower blood pressure. But 1 blood pressure medicine may not be enough for you. You may find the help you need with the 2 effective medicines in AZOR.

AZOR combines 2 effective medicines in 1 convenient pill.

Learn how each medicine in AZOR works in its own way to help lower blood pressure.

The medicine in AZOR

How it works

What it does

Angiotensin II receptor blocker (ARB)

Blocks a natural chemical in your body that causes blood vessels to narrow. This helps your blood vessels relax and widen.

Lowers

Your

Blood

pressure

Calcium channel blocker

Blocks the narrowing effect of calcium on your blood vessels. This helps your blood vessels relax.

http://www.AZOR.com/how_works.html

Section Two

Calcium-Channel Blockers: Drug Class and Indications

In Sudhof’s Lasker Award presentation he refers to the biochemical properties of synaptotagmin were found to precisely correspond to the extraordinary calcium-triggering properties of release, and to account for a regulatory pathway that also applies to other types of calcium-triggered fusion, for example fusion observed in hormone secretion. A CCB would have to block the calcium-triggering properties of release, and consequently, would block the release of neurohormones. This is because the fusion apparatus and its synaptotagmin-dependent control mechanism linked to the calcium channels, docking and priming synaptic vesicles, being blocked, disables the calcium-control of the vesicle fusion that is necessary for neurotransmitter release. Consequently, the end result would be increased vascular flow from the inhibition.

What are calcium channel blockers and how do they work?

In order to pump blood, the heart needs oxygen. The harder the heart works, the more oxygen it requires. Angina (heart pain) occurs when the supply of oxygen to the heart is inadequate for the amount of work the heart must do. By dilating the arteries, CCBs reduce the pressure in the arteries. This makes it easier for the heart to pump blood, and, as a result, the heart needs less oxygen. By reducing the heart’s need for oxygen, CCBs relieve or prevent angina. CCBs also are used for treating high blood pressure because of their blood pressure-lowering effects. CCBs also slow the rate at which the heart beats and are therefore used for treating certain types of abnormally rapid heart rhythms.

For what conditions are calcium channel blockers used?

CCBs are used for treating high blood pressure, angina, and abnormal heart rhythms (for example, atrial fibrillation, paroxysmal supraventricular tachycardia).

They also may be used after a heart attack, particularly among patients who cannot tolerate beta-blocking drugs, have atrial fibrillation, or require treatment for their angina.

Unlike beta blockers, CCBs have not been shown to reduce mortality or additional heart attacks after a heart attack.

CCBs are as effective as ACE inhibitors in reducing blood pressure, but they may not be as effective as ACE inhibitors in preventing the kidney failure caused by high blood pressure or diabetes.

They also are used for treating:

CCBs are also used in the prevention of migraine headaches.

Are there any differences among calcium channel blockers?

CCBs differ in their duration of action, the process by which they are eliminated from the body, and, most importantly, in their ability to affect heart rate and contraction. Some CCBs [for example, amlodipine (Norvasc)] have very little effect on heart rate and contraction so they are safer to use in individuals who have heart failure or bradycardia (a slow heart rate). Verapamil (Calan, Isoptin) and diltiazem (Cardizem) have the greatest effects on the heart and reduce the strength and rate of contraction. Therefore, they are used in reducing heart rate when the heart is beating too fast.

What are the side effects of calcium channel blockers?

The most common side effects of CCBs are constipation, nausea,headache, rash, edema (swelling of the legs with fluid), low blood pressure, drowsiness, and dizziness.
Liver dysfunction and over growth of gums may also occur. When diltiazem (Cardizem) or verapamil (Calan, Isoptin) are given to individuals with heart failure, symptoms of heart failure may worsen because these drugs reduce the ability of the heart to pump blood.
Like other blood pressure medications, CCBs are associated with sexual dysfunction.

http://www.medicinenet.com/calcium_channel_blockers/article.htm

Section Three

Brand and Generic Calcium Channel Blocking Agents

A drug may be classified by the chemical type of the active ingredient or by the way it is used to treat a particular condition. Each drug can be classified into one or more drug classes.

Calcium channel blockers block voltage gated calcium channels and inhibits the influx of calcium ions into cardiac and smooth muscle cells. The decrease in intracellular calcium reduces the strength of heart muscle contraction, reduces conduction of impulses in the heart, and causes vasodilatation.

Decrease in intracellular calcium in the heart decreases cardiac contractility. Decreased calcium in the vascular smooth muscle reduces its contraction and therefore causes vasodilatation.

Decrease in cardiac contractility decreases cardiac output and vasodilatation decreases total peripheral resistance, both of which cause a drop in blood pressure.

Calcium channel blocking agents are used to treat hypertension.

Filter by: — all conditions –AnginaAngina Pectoris ProphylaxisArrhythmiaAtrial FibrillationAtrial FlutterBipolar DisorderCluster HeadachesCoronary Artery DiseaseHeart FailureHigh Blood PressureHypertensive EmergencyHypertrophic CardiomyopathyIdiopathic Hypertrophic Subaortic StenosisIschemic StrokeMigraine PreventionNocturnal Leg CrampsPremature LaborRaynaud’s SyndromeSubarachnoid HemorrhageSupraventricular Tachycardia

Drug Name ( View by: Brand | Generic )

Afeditab CR (Pro, More…)generic name: nifedipine

Diltia XT (Pro, More…)generic name: diltiazem

Diltiazem Hydrochloride SR (More…)generic name: diltiazem

Nimotop (Pro, More…)generic name: nimodipine

Verelan PM (Pro, More…)generic name: verapamil

Cartia XT (Pro, More…)generic name: diltiazem

Adalat (More…)generic name: nifedipine

Calan SR (Pro, More…)generic name: verapamil

Cardizem (Pro, More…)generic name: diltiazem

Diltiazem Hydrochloride CD (More…)generic name: diltiazem

Isoptin SR (Pro, More…)generic name: verapamil

Nifediac CC (Pro, More…)generic name: nifedipine

Tiazac (Pro, More…)generic name: diltiazem

Procardia (Pro, More…)generic name: nifedipine

Adalat CC (Pro, More…)generic name: nifedipine

Cardizem LA (Pro, More…)generic name: diltiazem

Calan (Pro, More…)generic name: verapamil

Procardia XL (Pro, More…)generic name: nifedipine

Isoptin (More…)generic name: verapamil

Nifedical XL (Pro, More…)generic name: nifedipine

Plendil (Pro, More…)generic name: felodipine

Taztia XT (Pro, More…)generic name: diltiazem

Cardizem CD (Pro, More…)generic name: diltiazem

Norvasc (Pro, More…)generic name: amlodipine

Verelan (Pro, More…)generic name: verapamil

Cardene SR (Pro, More…)generic name: nicardipine

DynaCirc CR (Pro, More…)generic name: isradipine

Sular (Pro, More…)generic name: nisoldipine

Cardene (Pro, More…)generic name: nicardipine

Cardene IV (Pro, More…)generic name: nicardipine

Cleviprex (Pro, More…)generic name: clevidipine

Covera-HS (Pro, More…)generic name: verapamil

Dilacor XR (Pro, More…)generic name: diltiazem

Dilt-XR (Pro, More…)generic name: diltiazem

Diltiazem Hydrochloride XR (More…)generic name: diltiazem

Diltiazem Hydrochloride XT (More…)generic name: diltiazem

Diltzac (Pro, More…)generic name: diltiazem

Dynacirc (Pro, More…)generic name: isradipine

Matzim LA (Pro, More…)generic name: diltiazem

Nymalize (Pro, More…)generic name: nimodipine

Vascor (More…)generic name: bepridil

Section Four

Dysfunction of the Calcium Release Mechanism

For Disruption of Calcium Homeostasis in Vascular Smooth Muscle Cells, see

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

Part V: Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

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

http://pharmaceuticalintelligence.com/2013/08/26/heart-smooth-muscle-excitation-contraction-coupling-cytoskeleton-cellular-dynamics-and-ca2-signaling/

For Disruption of Calcium Homeostasis in Cardiomyocyte Cells, see

Aviva Lev-Ari, PhD, RN

Part VII: 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

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

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

http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Section Five

The Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

This topic is covered in

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/

Part V: Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

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

http://pharmaceuticalintelligence.com/2013/08/26/heart-smooth-muscle-excitation-contraction-coupling-cytoskeleton-cellular-dynamics-and-ca2-signaling/

Summary

Work on neurotransmitter release revealed a hierarchy of molecular machines that mediate the fusion of synaptic vesicles, the calcium-control of this fusion, and the embedding of calcium-controlled fusion in the context of the presynaptic terminal at the synapse. The neural transmission is described as a biological relay system. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers—neurotransmitters. When the calcium-controlled fusion at the presynaptic junction is blocked, as with a CCB, neurotransmitters are not released. The activity of the neurotransmitters would be to cause smaooth muscle contraction of the vessel. The CCB would cause relaxation and flow.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Part IX of this series of articles discussed the mechanism of the signaling of smooth muscle cells by the interacting parasympathetic neural innervation that occurs by calcium triggering neurotransmitter release by initiating synaptic vesicle fusion. It involves the interaction of soluble N-acetylmaleimide-sensitive factor (SNARE) and SM proteins, and in addition, the discovery of a calcium-dependent Syt1 (C) domain of protein- kinase C isoenzyme, which binds to phospholipids. It is reasonable to consider that it differs from motor neuron activation of skeletal muscles, mainly because the innervation is in the involuntary domain. The cranial nerve rooted innervation has evolved comes from the spinal ganglia at the corresponding level of the spinal cord. It is in this specific neural function that we find a mechanistic interaction with adrenergic hormonal function, a concept intimated by the late Richard Bing. Only recently has there been a plausible concept that brings this into serious consideration. Moreover, the review of therapeutic drugs that are used in blocking adrenergic receptors are closely related to the calcium-channels. Interesting too is the participation of a phospholipid bound protein-kinase isoenzyme C calcium-dependent domain Syt1. The neurohormonal connection lies in the observation by Katz in the 1950’s that the vesicles of the neurons hold and eject fixed amounts of neurotransmitters. The mechanism of this action will be futher discussed in Part X.

http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Posted in Calcium Signaling, Cell Biology, Signaling & Cell Circuits, Cytoskeleton, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, Origins of Cardiovascular Disease, tagged ATP, Aviva Lev-Ari, Calcium, Calcium in biology, Calcium-Channel Blocker, gene therapy, Heart Failure, Justin Pearlman, Larry H. Bernstein, Smooth muscle tissue on September 12, 2013| Leave a Comment »

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Author, Introduction: Larry H Bernstein, MD, FCAP

Author, Summary: Justin Pearlman, MD, PhD, FACC

and

Article Curator: Aviva Lev-Ari, PhD, RN

Article VIII Disruption of Calcium Homeostasis Cardiomyocytes and Vascular Smooth Muscle Cells The Cardiac and Cardiovascular Calcium Signaling Mechanism

Image created by Adina Hazan 06/30/2021

This article is the Part VIII in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells. Calcium has a storage and release cycle that flags activation of important cellular activities that include in particular the cycle activation of muscle contraction both to circulate blood and control its pressure and distribution. Homeostasis – the maintenance of status – requires controlled release of calcium from storage and return of calcium to storage. Such controls are critical both within cells, and for the entire biologic system. Thus the role of kidneys in maintaining the correct total body load of available calcium is just as vital as the subcellular systems of calcium handling in heart muscle and in the muscles that line arteries to control blood flow. The practical side to this knowledge includes not only identifying abnormalities at the cellular as well as system levels, but also identifying better opportunities to characterize disease and to intervene.

The Series consists of the following articles:

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

Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

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

http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

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

http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

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

Part V: Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

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

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Aviva Lev-Ari, PhD, RN

Part VII: 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

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

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

http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

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

http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: 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/

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This article has three Sections:

Section One:

Vascular Smooth Muscle Cells: The Cardiovascular Calcium Signaling Mechanism

Section Two:

Cardiomyocytes Cells: The Cardiac Calcium Signaling Mechanism

Section Three:

The Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Introduction

by Larry H Bernstein, MD, FACC

Introduction

This discussion in two Sections brings to a conclusion the two main aspects of calcium signaling and transient current induction in the cardiovascular system – involving vascular smooth muscle and cardiomyocyte. In this first Section, it extends the view of smooth muscle beyond the vascular smooth muscle to contraction events in gastrointestinal tract, urinary bladder, and uterus, but by inference, to ductal structures (gallbladder, parotid gland, etc.). This discussion also reinforces the ECONOMY of the evolutionary development of these functional MOTIFS, as a common thread is used again, and again, in specific contexts. The main elements of this mechanistic framework are:

the endoplasmic (sarcoplasmic) reticulum as a strorage depot for calcium needed in E-C coupling
the release of Ca(2+) into the cytoplasm
the generation of a voltage and current with contraction of the muscle cell unit
the coordination of smooth muscle cell contractions (in waves)
- this appears to be related to the Rho/Rho kinase pathway
there is also a membrane depolarization inherent in the activation mechanism
whether there is an ordered relationship between the calcium release and the membrane polarization, and why this would be so, in not clear
three different models of calcium release are shown from the MJ Berridge classification article below in Figure 1.
Model C is of special interest because of the focus on cytosolic (Ca+) ion transfers involving the interstitial cells of Cajal (Ramin e’ Cajal) through gap junctions

Santiago Ramón y Cajal (Spanish: [sanˈtjaɣo raˈmon i kaˈxal]; 1 May 1852 – 18 October 1934) was a Spanish pathologist, histologist and neuroscientist. He was awarded the Nobel Prize in Physiology or Medicine in 1906 together with Italian Camillo Golgi “in recognition of their work on the structure of the nervous system”. Relevant to this discussion, he discovered a new type of cell, to be named after him: the interstitial cell of Cajal (ICC). This cell is found interleaved among neurons embedded within the smooth muscles lining the gut, serving as the generator and pacemaker of the slow waves of contraction that move material along the gastrointestine, vitally mediating neurotransmission from motor nerves to smooth muscle cells . Cajal also described in 1891 slender horizontal bipolar cells in the developing marginal zone of lagomorphs.(See the Cajal’s original drawing of the cells) , considered by Retzius as homologues to the cells he found in humans and in other mammals (Retzius, 1893, 1894). The term Cajal–Retzius cell is applied to reelin-producing neurons of the human embryonic marginal zone.

Section One

Vascular Smooth Muscle Cells: The Cardiovascular Calcium Signaling Mechanism

Smooth Muscle Cell Calcium Activation Mechanisms

Michael J. Berridge

J Physiol 586.21 (2008) pp 5047–5061

Classification of Smooth Muscle Ca2+ Activation Mechanisms

Excitation–contraction coupling in SMCs occurs through two main mechanisms. Many SMCs are activated by Ca2+ signalling cascades (Haddock & Hill, 2005; Wray et al. 2005). In addition, there is a Rho/Rho kinase signaling pathway that acts by altering the Ca2+ sensitivity of the contractile system (Somlyo & Somlyo, 2003). Since the latter appears to have more of a modulatory function,most attention will focus on how Ca2+ signalling is activated. Since membrane depolarization is a key element for the activation of many SMCs,much attention will focus on the mechanisms responsible for depolarizing the membrane. However, there are other SMCs where activation depends on the periodic release of Ca2+ from internal stores. These different Ca2+ activation mechanisms fall into the following three main groups (Fig. 1).

Figure 1. The three main mechanisms responsible for generating the Ca2+ transients that trigger smooth

muscle cell (SMC) contraction

A, receptor-operated channels (ROCs) or a membrane oscillator induces the membrane depolarization (_V) that

triggers Ca2+ entry and contraction.

B, a cytosolic Ca2+ oscillator induces the Ca2+ signal that drives contraction.

C, a cytosolic Ca2+ oscillator in interstitial cells of Cajal (ICCs) or atypical SMCs induces the membrane depolarization

that spreads through the gap junctions to activate neighbouring SMCs. Reproduced from Berridge (2008), with permission

SOURCE for Figure 1: J Physiol 586.21 M. J. Berridge Smooth muscle cell calcium activation mechanisms 5048

Mechanism A.

Many SMCs are activated by membrane depolarization (_V) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). Themembrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction. The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is described in mechanism C.

Mechanism B.

The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Mechanism C.

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (_V) that triggers contraction through mechanism A. In the following sections, some selected SMC types will illustrate how these signalling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca2+ signals are activated.

Vascular, Lymphatic and Airway Smooth Muscle Cells

Vascular, lymphatic and airway smooth muscle, which generate rhythmical contractions over an extended period of time, have an endogenous pacemaker mechanism driven by a cytosolic Ca2+ oscillator. In addition, these SMCs also respond to neurotransmitters released from the neural innervation. In the case of mesenteric arteries, the perivascular nerves release both ATP and noradrenaline (NA). The ATP acts first to produce a small initial contraction that is then followed by a much larger contraction when NA initiates a series of Ca2+ transients (Lamont et al. 2003). Such agonist-induced Ca2+ oscillations are a characteristic feature of the activation mechanisms of vascular (Iino et al. 1994; Lee et al. 2001; Peng et al. 2001; Perez & Sanderson, 2005b; Shaw et al. 2004) and airway SMCs (Kuo et al. 2003; Perez & Sanderson, 2005a; Sanderson et al. 2008). In some blood vessels, a specific tone is maintained by the spatial averaging of asynchronous oscillations. However, there are some vessels where the oscillations in groups of cells are synchronized resulting in the pulsatile contractions known as vasomotion (Mauban et al. 2001; Peng et al. 2001; Lamboley et al. 2003; Haddock & Hill, 2005). Such vasomotion is also a feature of lymphatic vessels (Imtiaz et al. 2007). Another feature of this oscillatory activity is that variations in transmitter concentration are translated into a change in contractile tone through a mechanism of frequency modulation (Iino et al. 1994; Kuo et al. 2003; Perez & Sanderson, 2005a,b). Frequency modulation is one of the mechanisms used for encoding and decoding signalling information through Ca2+ oscillations (Berridge, 2007).

The periodic pulses of Ca2+ that drive these rhythmical SMCs are derived from the internal stores through the operation of a cytosolic Ca2+ oscillator (Haddock & Hill, 2005; Imtiaz et al. 2007; Sanderson et al. 2008). The following general model, which applies to vascular, lymphatic, airway and perhaps also to corpus cavernosum SMCs, attempts to describe the nature of this oscillator and how it can be induced or modulated by neurotransmitters. A luminal loading Ca2+ oscillation mechanism (Berridge & Dupont, 1994; Berridge, 2007) forms the basis of this cytosolic oscillator model that depends upon the following sequential series of events (Fig. 5).

Figure 5. Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release

of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave

The oscillator is induced/modulated by neurotransmitters such as acetylcholine (ACh), 5-hydroxytryptamine (5-HT),

noradrenaline (NA) and endothelin-1 (ET-1), which act through inositol 1,4,5-trisphosphate (InsP3) that initiates

the oscillatory mechanism. The sequence of steps 1–9 is described in the text. Reproduced from Berridge (2008),

with permission.

SOURCE for Figure 5: J Physiol 586.21 M. J. Berridge Smooth muscle cell calcium activation mechanisms 5053

http://pharmaceuticalintelligence.com/2013/08/26/heart-smooth-muscle-excitation-contraction-coupling-cytoskeleton-cellular-dynamics-and-ca2-signaling/

For Disruption of Calcium Homeostasis in Vascular Smooth Muscle Cells, see

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

Part V: Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

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

Section Two

Cardiomyocytes Cells: The Cardiac Calcium Signaling Mechanism

Cardiomyocytes and Ca2+ Channels

Published August 8, 2011 // JCB vol. 194 no. 3 355-365
The Rockefeller University Press, doi: 10.1083/jcb.201101100

Cellular mechanisms of cardiomyopathy

Pamela A. Harvey and
Leslie A. Leinwand

+Author Affiliations

Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309

Correspondence to Leslie Leinwand: leslie.leinwand@colorado.edu

Abbreviations

DCM dilated cardiomyopathy

HCM hypertrophic cardiomyopathy

MyH Cmyosin heavy chain

RCM restrictive cardiomyopathy

Introduction

The heart relies on a complex network of cells to maintain appropriate function. Cardiomyocytes, the contracting cells in the heart, exist in a three-dimensional network of endothelial cells, vascular smooth muscle, and an abundance of fibroblasts as well as transient populations of immune cells. Gap junctions electrochemically coordinate the contraction of individual cardiomyocytes, and their connection to the extracellular matrix (ECM) transduces force and coordinates the overall contraction of the heart. Intracellularly, repeating units of actin and myosin form the backbone of sarcomere structure, the basic functional unit of the cardiomyocyte (Fig. 1). The sarcomere itself consists of ∼20 proteins; however, more than 20 other proteins form connections between the myocytes and the ECM and regulate muscle contraction (Fig. 1 B). Given the complexity of the coordinated efforts of the many proteins that exist in multimeric complexes, dysfunction occurs when these interactions are disrupted.

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Figure 1. Anatomy of the cardiac sarcomere(A) Diagram of the basic organization of the sarcomere. The sarcomere forms the basic contractile unit in the cardiomyocytes of the heart. Thin filaments composed of actin are anchored at the Z line and form transient sliding interactions with thick filaments composed of myosin molecules. The M Line, I Band, and A Band are anatomical features defined by their components (actin, myosin, and cytoskeletal proteins) and appearance in polarized light. Titin connects the Z line with the M line and contributes to the elastic properties and force production of the sarcomere through its extensible region in the I Band. Coordinated shortening of the sarcomere creates contraction of the cardiomyocyte. (B) Representation of the major proteins of the cardiac sarcomere. Attachment to the ECM is mediated by costameres composed of the dystroglycan–glycoprotein complex and the integrin complex. Force transduction and intracellular signaling are coordinated through the costamere. The unique roles of each of these proteins are critical to appropriate function of the heart. T-cap, titin cap; MyBP-C, myosin-binding protein C; NOS, nitric oxide synthase.

Although the heart may functionally tolerate a variety of pathological insults, adaptive responses that aim to maintain function eventually fail, resulting in a wide range of functional deficits or cardiomyopathy. Although a multitude of intrinsic and extrinsic stimuli promote cardiomyopathies, the best described causes are the >900 mutations in genes expressed in the cardiomyocyte (Fig. 1 B; Wang et al., 2010). Mutations in most of these genes cause a diverse range of cardiomyopathies, many with overlapping clinical phenotypes. Mutations in sarcomeric genes are usually inherited in an autosomal-dominant manner and are missense mutations that are incorporated into sarcomeres (Seidman and Seidman, 2001). Thus far >400 mutations in 13 sarcomeric proteins including β-myosin heavy chain (β-MyHC), α-cardiac actin, tropomyosin, and troponin have been associated with cardiomyopathy (www.cardiogenomics.med.harvard.edu). Table I summarizes these mutated proteins.

Ca²⁺ regulation and calcineurin signaling

Ca²⁺ concentrations inside the cardiomyocyte are critically important to actin–myosin interactions. Ca²⁺ is sequestered within the sarcoplasmic reticulum and the sarcomere itself, which serves as an intracellular reserve that is released in response to electrical stimulation of the cardiomyocyte. After contraction, sarco/endoplasmic reticulum Ca²⁺-ATPase sequesters the Ca²⁺ back into the sarcoplasmic reticulum to restore Ca²⁺balance. There is a clear correlation between force production and perturbation of Ca²⁺regulation, alterations of which might directly induce pathological, anatomical, and functional alterations that lead to heart failure via activation of GPCRs (Minamisawa et al., 1999).

Ca²⁺ in the cytosol can be increased to modulate sarcomere contractility by signaling through Gα_q recruitment and activation of PLCβ. Ca²⁺ released from the sarcoplasmic reticulum activates calmodulin, which phosphorylates calcineurin, a serine/threonine phosphatase. Upon activation, calcineurin interacts with and dephosphorylates nuclear factor of activated T cells (NFAT), which then translocates into the nucleus. Calcineurin activation exacerbates hypertrophic signals and expedites the transition to a decompensatory state. Indeed, cardiac-specific overexpression of calcineurin or NFAT leads to significant cardiac hypertrophy that progresses rapidly to heart failure (Molkentin et al., 1998). Administration of antagonists of calcineurin attenuates the hypertrophic response of neonatal rat ventricular myocytes to stimuli such as phenylephrine (PE) and angiotensin II (Taigen et al., 2000).

Mechanotransduction and signaling in the cardiomyocyte

The responses of cardiomyocytes to systemic stress or genetic abnormalities are modulated by mechanosensitive mechanisms within the cardiomyocyte (Molkentin and Dorn, 2001; Seidman and Seidman, 2001; Frey and Olson, 2003). A complex network of proteins that connects the sarcomere to the ECM forms the basis of the mechanotransduction apparatus. For example, components of the costamere complex, which form the connection between the sarcomere and the ECM via integrins, initiate intracellular signaling and subsequently alter contractile properties and transcriptional regulation in response to membrane distortion. Mechanosensitive ion channels are also implicated in signal initiation in response to systemic stress (Le Guennec et al., 1990;Zhang et al., 2000; de Jonge et al., 2002). These channels are likely responsible for acute changes that might initiate other longer-term responses in the heart but are nonetheless important to consider when examining possible transducers of systemic and tissue alterations to the cardiomyocyte.

Changes in wall stress induce signaling pathways that are associated with the development of cardiac pathology. The many intracellular signaling pathways that mediate responses to increased demand on the heart have been extensively reviewed elsewhere (Force et al., 1999; Molkentin and Dorn, 2001; Heineke and Molkentin, 2006). Here, we focus on pathways that are intimately involved in pathogenesis (Fig. 4). Although their effects in compensatory responses early in pathology initially increase function by promoting growth and contractility, persistent responses eventually compromise function.

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Figure 4. Signaling pathways associated with cardiac hypertrophy.

Although many pathways are associated with cardiomyopathy, up-regulation of transcription and induction of apoptosis are major mediators of pathogenic responses in the heart. The GPCR-associated pathway (dark red) can be activated by ET-1 and AngII, which are released in response to reduced contractility, and mediates contractile adaptation through increased calcium release from the sarcoplasmic reticulum. Increased intracellular calcium activates calmodulin and induces activation of the transcription factor MEF2. Incorporation into the sarcomere of mutant proteins that exhibit reduced ATP efficiency inhibits the sequestration of calcium from the cytosol and further enhances increases in intracellular calcium concentration. GPCR signaling is also associated with activation of the Akt signaling pathway (light green) that induces fetal gene expression and the cardiac hypertrophic response through inhibition of GSK3β. Apoptotic pathways (light blue) are induced by cytochrome c (CytC) release from mitochondria and activation of death receptors (like FasR) by cytokines such as TNF. Calcium overload and myocyte loss significantly contribute to reduced contractility in many forms of cardiomyopathy. ET-1, endothelin-1; HDAC, histone deacetylase; NFAT, nuclear factor of activated T cells; MEF-2, myocyte enhancer factor 2; SERCA, sarco/endoplasmic reticulum calcium-ATPase; cFLIP, cellular FLICE-inhibitory protein; AngII, angiotensin II; FasR, Fas receptor.

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/

For Disruption of Calcium Homeostasis in Cardiomyocyte Cells, see

Aviva Lev-Ari, PhD, RN

Part VII: 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

Section Three

The Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

This topic is covered in

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

Summary

Justin D Pearlman, MD, PhD, FACC PENDING

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