Aleksandra Michalewska-Włudarczyk

Identification of mutational hot spots in LMNA encoding lamin A/C in patients with familial dilated cardiomyopathy

The familial form of dilated cardiomyopathy (DCM) occurs in about 20%–50% of DCM cases. It is a heterogenous genetic disease: mutations in more than 20 different genes have been shown to cause familial DCM. LMNA, encoding the nuclear membrane protein lamin A/C, is one of the most inportant disease gene for that disease. Therefore, we analyzed the LMNA gene in a large cohort of 73 patients with familial DCM. Clinical examination (ECG, echocardiography, and catheterization) was followed by genetic characterization of LMNA by direct sequencing. We detected five heterozygous missense mutations (prevalence 7%) in five different families characterized by severe DCM and heart failure with conduction system disease necessitating pacemaker implantation and heart transplantation. Four of these variants clustered in the protein domain coil 1B, which is important for lamin B interaction and lamin A/C dimerization. Although we identified two novel mutations (E203V, K219T) besides three known ones (E161K, R190Q, R644C), it was remarkable that four mutations represent LMNA hot spots. DCM patients with LMNA mutations show a notable homogenous severe phenotype as we could confirm in our study. Testing LMNA in such families seems to be recommended because genotype information in an individual could definitely be useful for the clinician.

A myomesin mutation associated with hypertrophic cardiomyopathy deteriorates dimerisation properties.

Myomesin plays an important structural and functional role in the M-band of striated muscles. The C-terminal domain 13 of myomesin dimerises and forms antiparallel strands which cross-link neighboring Myosin filaments and titin in the M-line of the sarcomeres. These interactions stabilise the contractile apparatus during striated muscle contraction. Since myomesin is an important component of the M-band we screened the myomesin gene for genetic variants in patients with hypertrophic cardiomyopathy (HCM). We identified the missense mutation V1490I in domain 12 of myomesin in a family with inherited HCM. Analytical ultracentrifugation experiments, circular dichroism spectra, and surface plasmon resonance spectroscopy of myomesin fragments were carried out to investigate the effects of the mutation V1490I on structure and function of myomesin domains 11-13 and 12-13. Both the wild type and mutated myomesin domains My11-13 revealed similar secondary structures and formed stable dimers. Mutated myomesin domains My11-13 and My12-13 dimers revealed a reduced thermal stability and a significantly decreased dimerisation affinity, showing disturbed functional properties of V1490I mutated myomesin. However, monomeric myomesin domains My11-12, i.e. without dimerisation domain 13 showed no difference in thermal stability between wild type and V1490I mutated myomesin. In conclusion, the V1490I mutation associated with HCM lead to myomesin proteins with abnormal functional properties which affect dimerisation properties of myomesin domain 13. These effects may contribute to the pathogenesis of HCM.

Effects of Trans-Endocardial Delivery of Bone Marrow-Derived CD133+ Cells on Left Ventricle Perfusion and Function in Patients With Refractory Angina: Final Results of Randomized, Double-Blinded, Placebo-Controlled REGENT-VSEL Trial.

Rationale: New therapies for refractory angina are needed. Objective: Assessment of transendocardial delivery of bone marrow CD133+ cells in patients with refractory angina. Methods and Results: Randomized, double-blinded, placebo-controlled trial enrolled 31 patients with recurrent Canadian Cardiovascular Society II–IV angina, despite optimal medical therapy, ≥1 myocardial segment with inducible ischemia in Tc-99m SPECT who underwent bone marrow biopsy and were allocated to cells (n=16) or placebo (n=15). Primary end point was absolute change in myocardial ischemia by SPECT. Secondary end points were left ventricular function and volumes by magnetic resonance imaging and angina severity. After 4 months, there were no significant differences in extent of inducible ischemia between groups (summed difference score mean [±SD]: 2.60 [2.6] versus 3.63 [3.6], P=0.52; total perfusion deficit: 3.60 [3.6] versus 5.01 [4.3], P=0.32; absolute changes of summed difference score: −1.38 [5.2] versus −0.73 [1.9], P=0.65; and total perfusion deficit: −1.33 [3.3] versus −2.19 [6.6], P=0.65). There was a significant reduction of left ventricular volumes (end-systolic volume: −4.3 [11.3] versus 7.4 [11.8], P=0.02; end-diastolic volume: −9.1 [14.9] versus 7.4 [15.8], P=0.02) and no significant change of left ventricular ejection fraction in the cell group. There was no difference in number of patients showing improvement of ≥1 Canadian Cardiovascular Society class after 1 (41.7% versus 58.3%; P=0.68), 4 (50% versus 33.3%; P=0.63), 6 (70% versus 50.0%; P=0.42), and 12 months (55.6% versus 81.8%; P=0.33) and use of nitrates after 12 months. Conclusion: Transendocardial CD133+ cell therapy was safe. Study was underpowered to conclusively validate the efficacy, but it did not show a significant reduction of myocardial ischemia and angina versus placebo. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01660581.

Exercise-induced mobilisation of endothelial progenitor cells in patients with premature coronary heart disease

BACKGROUND Endothelial progenitor cells (EPC) derive from bone marrow and participate in both endothelial regeneration and development of new blood vessels. EPC also play a role in the atherosclerotic process, and their number correlates negatively with the presence of classical risk factors. AIM To evaluate circulating EPC count and their exercise-induced mobilisation in patients with premature coronary artery disease (CAD). METHODS The study group included 60 patients with stable CAD diagnosed before 45 years of age. The control group consisted of 33 healthy age- and gender-matched volunteers. Venous blood was sampled 3 times in order to assess circulating EPC count immediately before an exercise test (EPC 0) and at 15 min (EPC 15) and 60 min (EPC 60) after the exercise test. RESULTS Circulating EPC count in the study group at rest and at 15 min after exercise was comparable (2.1 vs. 2.1 cell/μL, p = 0.35) and increased significantly at 60 min after exercise in comparison to resting values (2.1 vs. 3.2 cell/μL, p < 0.00001). In the control group, circulating EPC count increased significantly at 15 min after exercise (2.0 vs. 3.5 cell/μL, p < 0.0001) but later decreased at 60 min after exercise, although it remained greater than at rest (2.7 vs. 2.0 cell/μL, p < 0.0002). Circulating EPC count at rest and at 60 min after exercise was comparable in the two groups (2.1 vs. 2.0 cell/μL, p = 0.96; and 3.2 vs. 2.7 cell/μL, p = 0.13, respectively) but it was significantly lower in the study group compared to the control group at 15 min after exercise (2.1 vs. 3.5 cell/μL, p < 0.00001). Circulating EPC count at rest and at 15 min after exercise did not correlate with the number of stenosed coronary arteries but at 60 min after exercise it was greater in patients with one-vessel disease compared to those with two- or three-vessel disease (4.2 vs. 3.4 cell/μL, p = 0.01; and 4.2 vs. 2.3 cell/μL, p = 0.00003). However, no difference in circulating EPC count was seen at 60 min after exercise between patients with two- or three-vessel disease (3.4 vs. 2.3 cell/μL, p = 0.3). CONCLUSIONS 1. Circulating EPC count at rest is comparable between subjects with premature atherosclerosis and healthy volunteers. 2. A single bout of physical exercise causes a significant increase in circulating EPC count in both groups, but the dynamics of exercise-induced EPC mobilisation is different, with delayed exercise-induced EPC mobilisation in subjects with premature CAD. 3. The extent of atherosclerotic coronary lesions does not influence circulating EPC count at rest.

Effects of Transendocardial Delivery of Bone Marrow–Derived CD133 + Cells on Left Ventricle Perfusion and Function in Patients With Refractory Angina

Rationale: New therapies for refractory angina are needed. Objective: Assessment of transendocardial delivery of bone marrow CD133+ cells in patients with refractory angina. Methods and Results: Randomized, double-blinded, placebo-controlled trial enrolled 31 patients with recurrent Canadian Cardiovascular Society II–IV angina, despite optimal medical therapy, ≥1 myocardial segment with inducible ischemia in Tc-99m SPECT who underwent bone marrow biopsy and were allocated to cells (n=16) or placebo (n=15). Primary end point was absolute change in myocardial ischemia by SPECT. Secondary end points were left ventricular function and volumes by magnetic resonance imaging and angina severity. After 4 months, there were no significant differences in extent of inducible ischemia between groups (summed difference score mean [±SD]: 2.60 [2.6] versus 3.63 [3.6], P=0.52; total perfusion deficit: 3.60 [3.6] versus 5.01 [4.3], P=0.32; absolute changes of summed difference score: −1.38 [5.2] versus −0.73 [1.9], P=0.65; and total perfusion deficit: −1.33 [3.3] versus −2.19 [6.6], P=0.65). There was a significant reduction of left ventricular volumes (end-systolic volume: −4.3 [11.3] versus 7.4 [11.8], P=0.02; end-diastolic volume: −9.1 [14.9] versus 7.4 [15.8], P=0.02) and no significant change of left ventricular ejection fraction in the cell group. There was no difference in number of patients showing improvement of ≥1 Canadian Cardiovascular Society class after 1 (41.7% versus 58.3%; P=0.68), 4 (50% versus 33.3%; P=0.63), 6 (70% versus 50.0%; P=0.42), and 12 months (55.6% versus 81.8%; P=0.33) and use of nitrates after 12 months. Conclusion: Transendocardial CD133+ cell therapy was safe. Study was underpowered to conclusively validate the efficacy, but it did not show a significant reduction of myocardial ischemia and angina versus placebo. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01660581.

Effects of trans-endocardial delivery of bone marrow-derived CD133+ cells on angina and quality of life in patients with refractory angina: A sub-analysis of the REGENT-VSEL trial

BACKGROUND The REGENT-VSEL trial demonstrated a neutral effect of transendocardial injection of autologous bone marrow (BM)-derived CD133+ in regard to myocardial ischemia. The current sub-analysis of the REGENT VSEL trial aims to assess the effect stem cell therapy has on quality of life (QoL) in patients with refractory angina. METHODS Thirty-one patients (63.0 ± 6.4 years, 70% male) with recurrent CCS II-IV angina, despite optimal medical therapy, enrolled in the REGENT-VSEL single center, randomized, double-blinded, and placebo-controlled trial. Of the 31 patients, 16 individuals were randomly assigned to the active stem cell group and 15 individuals were randomly assigned to the placebo group on a 1:1 basis. The inducibility of ischemia, (≥ one myocardial segment) was confirmed for each patient using Tc-99m SPECT. QoL was measured using the Seattle Angina Questionnaire. Each patient completed the questionnaire prior to treatment and at the time of their outpatient follow-up visits at 1, 4, 6, and 12 months after cell/placebo treatment. RESULTS The main finding of the REGENT-VSEL trial sub-analysis was that transendocardial injection of autologous BM-derived CD133+ stem cells in patients with chronic refractory angina did not show significant improvement in QoL in comparison to the control group. Moreover, there was no significant difference between cell therapy and placebo in a number of patients showing improvement of at least 1 Canadian Cardiovascular Society class during the follow-up period. CONCLUSIONS Intra-myocardial delivery of autologous CD133+ stem cells is safe and feasible but does not show a significant improvement in the QoL or angina pectoris symptoms in patients with chronic myocardial ischemia.

Effects of Transendocardial Delivery of Bone Marrow–Derived CD133+ Cells on Left Ventricle Perfusion and Function in Patients With Refractory AnginaNovelty and Significance: Final Results of Randomized, Double-Blinded, Placebo-Controlled REGENT-VSEL Trial

Rationale: New therapies for refractory angina are needed. Objective: Assessment of transendocardial delivery of bone marrow CD133+ cells in patients with refractory angina. Methods and Results: Randomized, double-blinded, placebo-controlled trial enrolled 31 patients with recurrent Canadian Cardiovascular Society II–IV angina, despite optimal medical therapy, ≥1 myocardial segment with inducible ischemia in Tc-99m SPECT who underwent bone marrow biopsy and were allocated to cells (n=16) or placebo (n=15). Primary end point was absolute change in myocardial ischemia by SPECT. Secondary end points were left ventricular function and volumes by magnetic resonance imaging and angina severity. After 4 months, there were no significant differences in extent of inducible ischemia between groups (summed difference score mean [±SD]: 2.60 [2.6] versus 3.63 [3.6], P=0.52; total perfusion deficit: 3.60 [3.6] versus 5.01 [4.3], P=0.32; absolute changes of summed difference score: −1.38 [5.2] versus −0.73 [1.9], P=0.65; and total perfusion deficit: −1.33 [3.3] versus −2.19 [6.6], P=0.65). There was a significant reduction of left ventricular volumes (end-systolic volume: −4.3 [11.3] versus 7.4 [11.8], P=0.02; end-diastolic volume: −9.1 [14.9] versus 7.4 [15.8], P=0.02) and no significant change of left ventricular ejection fraction in the cell group. There was no difference in number of patients showing improvement of ≥1 Canadian Cardiovascular Society class after 1 (41.7% versus 58.3%; P=0.68), 4 (50% versus 33.3%; P=0.63), 6 (70% versus 50.0%; P=0.42), and 12 months (55.6% versus 81.8%; P=0.33) and use of nitrates after 12 months. Conclusion: Transendocardial CD133+ cell therapy was safe. Study was underpowered to conclusively validate the efficacy, but it did not show a significant reduction of myocardial ischemia and angina versus placebo. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01660581.