Cardiac model systems and therapies

Question 1

What is VLCAD deficiency?

Answer 1

A rare mitochondrial disease with a mutation in the ACADVL gene, which normally produces an enzyme called very-long-chain acyl-CoA dehydrogenase (VLCAD). This enzyme is important in the fatty acid oxidation process.

Question 2

Explain what happens when the VLCAD enzyme is deficient.

Answer 2

Normally, VLCAD is responsible for the conversion of long-chain acyl-CoA into enoyl-CoA. Following the normal metabolic pathway, this would ultimately result in the production of ATP.
If VLCAD is deficient, this means that enoyl-CoA cannot be produced and that acyl-CoA accumulates. Since the upstream enzymes of VLCAD are still active, acyl-CoA is coverted back into acylcarnitine by the upstream enzymes. This results in:
- energy shortage (since ATP can no longer be produced)
- accumulation of acylcarnitines

Question 3

What are clinical characteristics of VLCAD deficiency?

Answer 3

• Liver hypoglycemia (low blood sugar level)
• Cardiomyopathy and/or arrythmia (heart)
• Myopathy of the muscle (muscle weakness) and at least two of the following: myoglobulinuria, myalgia, exercise intolerance, muscle weakness, and/or frequent fatigue.

Question 4

What can trigger symptoms in VLCAD deficient patients?

Answer 4

Symptoms are often triggered during catabolism (fasting, activity, infection), which can be fatal.

Question 5

Since 2007, VLCAD deficiency is included in the newborn screening (hielprik).
What are pros and cons of including VLCAD deficiency in the newborn screening?

Answer 5

Pros:
- Prevent early-life complications
- Know all Dutch patients and can use their cells

Cons:
- Cannot prevent late-life complications
- Cannot predict future disease (and how the disease will develop (mild/severe))

Question 6

What physiological process can be used to study the pathophysiology of VLCAD deficiency?

Answer 6

(Long-chain) fatty acid oxidation (FAO) flux

Question 7

What is seen in patients with VLCAD deficiency in regard to FAO-flux?

Answer 7

• That the FAO flux is variable between patients and can be increased when culturing at 30ºC.
• That the FAO flux correlates with clinical severity.

Question 8

Fill in the sentences.

• A low or high FAO flux correlates with a high clinical severity score.
• A low or high FAO flux correlates with a low clinical severity score.

Answer 8

• A low FAO flux correlates with a high clinical severity score.
• A high FAO flux correlates with a low clinical severity score.

Question 9

Resveratrol and bezafibrate are two (experimental) drugs (for VLCAD deficiency). What is seen when these drugs are given to VLCAD deficient patient fibroblasts?

Answer 9

They increase FAO flux in long chain fatty acid oxidation of patient fibroblasts.

Question 10

What fuels can be/are used by the body?

Answer 10

• Glucose
• Fat
• Protein/amino acids
• Ketones

Question 11

What kind of fuel increases in plasma level upon starvation?

Answer 11

Ketones

Question 12

What is the function of ketones?

Answer 12

They are an alternative and glucose-sparing fuel source.

Question 13

What is believed to be a problem in VLCAD deficient patients in regard to the use of fuel source?

Answer 13

VLCAD patients have a low concentration of ketones in their blood. This is because the burning of fat (beta-oxidation) is required to make ketones. Since VLCAD patients cannot even begin to oxidize fat, their hypoglycemia comes without ketones (hypoketotic hypoglycemia).

Question 14

A study used a certain method to measure the energy balance during exercise. What did they do and what was seen?

Answer 14

They constructed a 31P magnetic resonance spectroscopy, where the patient was able to exercise while lying in the MRI. What was seen was that compared to healthy subjects, the VLCAD deficient patients had decreased phosphocreatine and increased phosphate levels in their blood (increased Pi/PCr ratio).

Question 15

To study ketones as an alternative energy source, ketone esters (KE) or carbohydrate (as control) were adminstered to test subjects. Subjects where then asked to work out on a regular exercise bike and afterwards bike 10 minutes on the MRI bike.
What were differences that got detected in regard to the administration of KE compared to the carbohydrates?

Answer 15

• Ketones induce ketosis, whereas carbohydrates don’t.
• Ketones lower plasma acylcarnitines comared to carbohydrates.
• Ketones reduce Pi/PCr ratio → suggestive of improved ATP homeostasis

Question 16

In another study, human induced pluripotent stemcell were used and induced to cardiomyocytes as a model for the arrhythmias associated with VLCAD deficiency. What was confirmed through the use of this iPSCs model?

Answer 16

The biochemical phenotype of VLCAD deficiency

Question 17

In the same iPSCs study where human induced pluripotent stem cells were reprogrammed into cardiomyocytes with a VLCAD deficiency, the researchers also looked at the cardiac action potentials.
What was seen in the cardiomyocytes with a VLCAD deficiency compared to controls?

Answer 17

That in the VLCAD deficient cardiomyocytes, the action potential declines more rapidly compared to controls (i.e. action potential is less sustained or shortened).

Question 18

In the same iPSCs study where human induced pluripotent stem cells were reprogrammed into cardiomyocytes with a VLCAD deficiency, the researchers also looked at the cardiac action potentials.
Besides the shortened action potentials, what can be seen in this picture?

Answer 18

Delayed after depolarization (DADs) in VLCAD deficient cardiomyocytes. DADs arise from the resting potential after full repolarization of an action potential and may reach threshold for activation.

Question 19

In the same human iPSCs study with reprogrammed cardiomyocytes, the researchers wanted to know the cause of the observed DADs in VLCAD deficient cardiomyocytes. What was seen?

Answer 19

Accumulation of systolic and diastolic intracellular calcium

Question 20

What is seen when resveratrol is given to VLCAD deficient patients?

Answer 20

Resveratrol is able to reduce acylcarnitine accumulation, to rescue the action potential, to rescue the amount of DADs and intracellular calcium. However, in the study explained in the lecture, resveratrol is only able to rescue the phenotype in one of two patients.

Question 21

On what enzyme does resveratrol act on in the fatty acic oxidation process?

Answer 21

On the downstreams VLCAD enzyme.

Question 22

If inhibition of the downstreams VLCAD enzyme does not work, what other option is there?

Answer 22

Inhibiting the fatty acid oxidation cycle (with etomoxir)

Question 23

What is seen when etomoxir is given to VLCAD deficient patients in regard to:
- acylcarnitine accumulation
- lenght of action potential
- DADs and intracellular calcium

Answer 23

• acylcarnitine accumulation → etomoxir prevents acylcarnitine accumulation
• lenght of action potential → etomoxir prolongs action potentials
• DADs and intracellular calcium →attenuates DADs and intracellular calcium

All processes are rescues in all patients.

Question 24

Name three factors that trigger disorders of mitochondrial long-chain fatty acid oxidation.

Answer 24

• Nutrition and diet adherence
• Exercise
• Inflammatory triggers

Question 25

Dermal fibroblasts (MADD and VLCADD) are incubated for 24 hours with lipopolysaccharide (LPS), where subsequently cytokine expression was measured with the use of ELISA.
What was seen in controls versus severe and mild VLCADD and MADD?

Answer 25

• In VLCADD, IL-6 expression was reduced tremendeously.
• In MADD, IL-6 expression was completely absent.

Question 26

Take home messages