Molecular basis of dilated cardiomyopathy

Prasad Gunaruwan

21st June 2011

Introduction

• Didactic, regular questions to the audience

• Pathology of dilated cardiomyopathy

• Myocyte apoptosis, regulators

• Structure of the cardiac myocyte

• Inherited DCM and mechanisms

• Extra-cellular components in DCM

• Therapeutic potential

Classification of DCM

• NEJM pic

N Engl J Med 2011;364:1643-56.

Pathology of DCM

N Engl J Med 2011;364:1643-56.

Pathology of DCM

• LV dilatation

• Systolic dysfunction

• Myocyte death and fibrosis

• Regardless of etiology this is the end result

Pathology of DCM

Systolic dysfunction

LV Dilatation Myocyte death and fibrosis

Cardiac Myocyte Apoptosis

• Myocyte death as a result of necrosis, autophagy (self destruction and recycling of raw material) and apoptosis (programmed cell death)

• Apoptosis mediated via external pathway (death ligands binding to cell surface receptors) and internal pathway (via mitochondria and ER)

• Internal pathway has many stimuli (hypoxia, DNA damage, radiation, loss of trophic factors, toxins…)

• Apoptosis signals kept in check at different levels in the activation cascade

J. Clin. Invest. 115:565–571 (2005).

Question to..last row furthest from the door……(ususally Dr Hayes)

• What is not a pro-apoptotic intra-cellular component?

a)Active caspase 3

b)Active caspase 9

c)Active caspase 12

d)Active caspase 8

e)X linked Inhibitor of Apoptosis (XIAP)

Evidence for increased apoptosis in DCM

• Apoptosis ‘measured’ by caspase activation, mRNA levels of pro-apoptotic components (Bcl-2, Bax, Bcl-xL) and activation of their gene encoding by p-53.

• Control apoptosis rate 0.001-0.002% vs 0.08-0.25% in failing hearts.

• Loss of myocytes is not debated, but the contribution of apoptosis is controversial.

• Causal relationship in mouse models and correlations in human studies

Mouse Model: apoptosis causes DCM

• Transgenic mice, cardiac restricted pro-caspase 8 fusion protein expression.

• The molecule dimerization and activation could be switched on by a drug.

• Within hours of activation death with severe myocyte damage.

These had low levels of caspase and apoptosis (0.023% vs 0.002%).DCM partially arrested with caspase inhibition.

J. Clin. Invest. 111:1497–1504 (2003)

Human Studies: apoptosis in DCM (not causal)

• 4 clinical groups (normal volume status, AR with preserved EF, AR with reduced EF, severe DCM/AR)

• LV biopsies and assay for apoptosis

• Patients were on medications

Caspase levels in DCM

Am J Cardiol 2009;103:1261–1268)

Other Pro-apoptotic markers

Ischaemia and apoptosis

Am J Physiol Heart Circ Physiol280: H2313–H2320, 2001.

Other etiology to apoptosis links • Ang-II

– Gq transduces Ang-II signals– Gαq (over expression of α subunit) predisposes to

transcriptional activation of BH3 like protien Nix/Gp 130/STAT

– Eventually Bcl-xL and apoptosis

• Severe activation of Nix in pregnant mice: fulminant DCM, inhibitors of Nix prevent the DCM.

• Myocardial stretch promotes Titin iso-type switching and p53 activation, which promotes encoding for pro-apoptotic molecules

Question to… Second row furthest from the door.. (usually Prof Fletcher)

• Which of the following DCM etiology is yet to have a identified link to apoptosis?

a)Ischaemic cardiomyopathy

b)Peripartum cardiomyopathy

c)Severe aortic regurgitation

d)Persistantly activated Renin-Ang-Ald system

e)Alcohol induced DCM

Summary so far…

• DCM: LV dilatation, myocyte death/fibrosis, systolic dysfunction.

• Multiple etiologies somehow converge on the above final status.

• Myocyte apoptosis rates higher in severe DCM.• Some data suggest apoptosis causally linked to

DCM.• Some possible pathways to link acquired DCM

etiology to apoptosis.

Inherited DCM

• Genetic defects resulting in structural defects within the sarcomere

• A brief review of normal sarcomeric structure

Sarcomere structure

Lamin A/C DCM

• Lamins are ‘structural’ proteins located within the nuclear side of the nuclear membrane

• How they cause DCM is still being studied

Structure of Lamins

Lamins and the nuclear envelope

Lamins: structural vs functional

• Important molecules to maintain nuclear envelope structure.

• Lamin A has intra-nuclear components that regulates nuclear replication, gene expression and regulates transcription of RNA.

• Mutated Lamin A: mis-localises Emerin to ER, and Lamin C to the nucleoplsm.

• Mis-localisation of Lamin C into the nucleoplsm seems to be key in DCM.

• Mouse models show link to extra-cellular kinase activation

Lamins and Extra-cellular Kinases

• Lamin mutation knock-in mice noted to have increased ERK activity prior to DCM

• Drugs inhibiting ERK activity delayed DCM onset and severity

• ? Lamin mutations may allow RNA transcriptions of these kinases

• ? Normal lamins provide substrate for the kinases

Human Molecular Genetics, 2009, Vol. 18, No. 2 241–247

Question to.. First row furthest from door.. (usually Dr Collins..)

What is incorrect?a) Lamin A/C mutations lead to AutoD and AutoR disorders.

b) Lamins are important structural proteins in the sarcolemma.

c) Lamin mutation may promote extra-cellular kinase activation.

d) Lamins maintain the structure of the nuclear envelope.

e) Mis-localisation of Lamin C is associated with DCM.

Summary so far..

• DCM: LV dilatation, systolic dysfunction, myocyte death and fibrosis.

• Apoptosis may be a key feature in loss of myocytes.

• Links between etiologies and apoptosis.

• Any structural protein abnormality in the sarcolemma can lead to DCM.

• Lamins are not just structural proteins

What about the extra-cellular matrix?

Changes in the ECM with DCM

• Increased collagen turn-over, ratio of C I:III reduced in DCM

• Mast cells and fibroblast activation, increased pro-fibrotic cytokines and growth factors

• Loss of collagen cross-linking, mural re-alignment (‘slippage’)

• Activated Matrix-Metalloproteinases.

Matrix Metallo-Proteinases (MMP)

MMP and TIMP

• TIMPs are Tissue Inhibitors of MMPs

• Ratio between MMP: TIMP in CHF favours persistant activation of MMPs

• MMP inhibitors of benefit in mouse models of MI and LVF

• Led to the trial of MMP inhibitor in Post STEMI LVF: negative result, blamed on inadequate dosing of the MMP inhibitor.

Summary so far..

• DCM: LV dilatation, myocyte death and fibrosis, systolic dysfunction.

• Apoptosis a key in myocyte death, could be causal in DCM

• Any structural protein abnormality can lead to DCM

• Extra cellular matrix changes also contribute to LV dilatation and systolic dysfunction

What about cardiac stem cells?

• Not a lot about what happens to CPC in DCM

• Presume they are also affected by the etiological factors and down-stream cascades

• Anthracycline induced DCM: Injection of CPC harvested prior to exposure reduce effects of DCM

Therapeutic Potential

• Myriad of potential therapeutic targets.

• Apoptosis work > 10 yrs, no clinical trials yet.

• Some trials on ECM have failed.

• ? Kinase inhibitors for lamin DCM

Thank you