BIOEN345

Failure Analysis
of Human
Physiology
Sonette Steczina
2020 April 15
 Contractile machinery of the heart
• Actin “slides” along myosin to
generate contraction
• Myosin motor hydrolyzes ATP in
the process
• Tightly regulated process by
troponin complex and myosin
binding protein-C (MyBP-C)
MYOSIN • Can interact with both myosin
and actin filament
• Regulates how available thin
filament is for myosin
attachment
Harris S. et al., 2011
This can result in excessive contractility
if destabilized by disease state

https://veteriankey.com/electrical-activity-of-the-heart/ Carrier L. et al., 2015

 Hypertrophic Cardiomyopathy (HCM)
• HCM is a non-ischemic cardiomyopathy
• Affecting ~0.2% of the world’s population
• Determined to be a major cause of sudden cardiac death in the young
• Phenotypically leads to thickening of the ventricular muscle walls of
the heart
• Strongly associated with familial genetic mutations in proteins of the
muscle sarcomere

How physiology is disrupted by disease causing genetic mutations and

how it is studied?

Kayashi T., 2020

 Florence HCM cohort: Increased prevalence of MyBP-C
mutations

General population
TNNT2
TPM1
Complex TNNI2

MYL-2
MYBPC3- non E258K
MYBPC3-E258K
MYH7
MYL-2
MYH7 (30%) MYBPC3 (54%)
Complex
TPM1
TNNT2
TNI
 Florence HCM cohort: Increased prevalence of MyBP-C
mutations
TUSCANY
Florence, Italy

General population
TNNT2
TPM1
Complex TNNI2

MYL-2
MYBPC3- non E258K
MYBPC3-E258K
MYH7
MYL-2
MYH7 (30%) MYBPC3 (54%)
Complex HCM Florence cohort
TPM1
TNNT2 Other
TNI 11% Other MYBPC3
37%
31%

21%
MYH7

MYBPC3-E258K
Rare access to clinical tissue samples from myectomy
• Clinical samples were collected from a subset of recruited patients with
E258K mutation in MyBP-C
• Biopsy samples can be collected during myectomy surgery

However…

• Access to human patient tissue is limited

• Results from these samples are indicative of late-stage in the disease

It is of great interest in research to engineer physiologically relevant disease

models to study the disease and potential therapeutics in vitro
Pluripotent stem cells = Invaluable disease modeling tool
Starting Material

Embryonic stem cells

Obtained from embryo

Induced pluripotent stem cells

Can be engineered from

somatic cell source

Adapted from: https://www.ddw-online.com/drug-discovery/p142795-induced-pluripotent-stem-cells:-a-model-for-transforming-drug-discovery.html

Pluripotent stem cells = Invaluable disease modeling tool
Starting Material Use in the laboratory

Embryonic stem cells

Obtained from embryo Self-renewal

Differentiation

Induced pluripotent stem cells

Can be engineered from

somatic cell source

Adapted from: https://www.ddw-online.com/drug-discovery/p142795-induced-pluripotent-stem-cells:-a-model-for-transforming-drug-discovery.html

 Reprogramming to generate a patient specific
stem cell line
• 2006: First induction of human fibroblast cells to induced pluripotent
stem cells (iPSCs)
• Retroviral transduction of 4 transcription factors (Oct4, Sox2, Klf4, c-Myc)
Now use non-integrating Sendai virus as delivery method
 Reprogramming to generate a patient specific
stem cell line
• 2006: First induction of human fibroblast cells to induced pluripotent
stem cells (iPSCs)
• Retroviral transduction of 4 transcription factors (Oct4, Sox2, Klf4, c-Myc)
Now use non-integrating Sendai virus as delivery method

• 3 – 4 weeks to reprogram peripheral blood mononuclear cells (PBMC) to iPSCs

HCM patient
(E258K mutation)
Peripheral Isolation of
Selection of viable clones iPSCs
blood PBMC’s +Oct4, Sox2, Klf4, c-Myc
Quality control to ensure engineered iPSCs have
properties of stemness
Sequencing of
Characterization Expansion Differentiation
viable clones
Quality control to ensure engineered iPSCs have
properties of stemness
Sequencing of
Characterization Expansion Differentiation
viable clones

A) Karyotyping

B) Pluripotency markers C) Differentiation to all 3 germ layers

Fu S. et al., 2018
Quality control to ensure engineered iPSCs have
properties of stemness
Sequencing of
Characterization Expansion Differentiation
viable clones

A) Karyotyping
B) Pluripotency markers Self-renewal

C) Differentiation to all
3 germ layers
Quality control to ensure engineered iPSCs have
properties of stemness
Sequencing of
Characterization Expansion Differentiation
viable clones

A) Karyotyping
B) Pluripotency markers Self-renewal

C) Differentiation to all
3 germ layers

93.5% purity for

cardiomyocytes
Using pluripotent stem cells to generate a patient specific
disease model in-a-dish
• Differentiate patient pluripotent stem cells into cardiomyocytes
• Will beat spontaneously in the dish as a cell sheet
• Will still have the E258K mutation in myosin binding protein-C

HCM patient
(E258K mutation)
iPSCs-derived
iPSCs Cardiomyopathy model
cardiomyocytes
with E258K mutation
Reprogramming for
pluripotency induction Differentiation
 How can this iPSC-CM, disease in-a-dish tool
be used in the laboratory?
Whole organ &
Multicellular
 Force measurements
 Pathology staining We want to determine
the disease
mechanism at the cell
Single Cells
and subcellular scale
 Contraction and relaxation
 Calcium transients
in order to better
 Cell morphology
understand the
disease at the whole
organ scale
Subcellular
 RNA/protein expression
 Myofibrils mechanics
https://veteriankey.com/electrical-activity-of-the-heart/
 Measurement of cell contraction and relaxation
IonOptix Analysis

Cell Length

Sarcomere
Single Cells length
 Contraction and relaxation
 Calcium transients
 Cell morphology

Ca2+ transient

https://veteriankey.com/electrical-activity-of-the-heart/
 Assessment of cell morphology
Immunohistochemistry

Cell sheet

Nucleus
Single Cells
 Contraction and relaxation
 Calcium transients
 Cell morphology

Single iPSC-
cardiomyocyte

Troponin T
staining
myofibrils
https://veteriankey.com/electrical-activity-of-the-heart/
 Model system to test candidate therapeutics
Discovery of Pre-clinical
novel biomarkers drug screening

Cardiomyopathy model
with E258K mutation

Single Cells
 Contraction and relaxation
 Calcium transients
 Cell morphology

CRISPR/Cas9 Viral gene

genome editing therapies

https://veteriankey.com/electrical-activity-of-the-heart/
 Model system to test candidate therapeutics
Discovery of Pre-clinical
novel biomarkers drug screening

Cardiomyopathy model
with E258K mutation

Single Cells
 Contraction and relaxation
 Calcium transients
 Cell morphology

CRISPR/Cas9 Viral gene

genome editing therapies
Overexpression
of proteins

https://veteriankey.com/electrical-activity-of-the-heart/
 Model system to test candidate therapeutics
Discovery of Pre-clinical
novel biomarkers drug screening

Cardiomyopathy model
with E258K mutation

Single Cells
 Contraction and relaxation
 Calcium transients
 Cell morphology dATP as a
myosin activator

CRISPR/Cas9 Viral gene

genome editing therapies
Overexpression
of proteins

https://veteriankey.com/electrical-activity-of-the-heart/
Turning it over to Matt 