Dataset Information

Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

ABSTRACT:

Background and purpose

Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV 1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling.

Experimental approach

Using the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV 1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS.

Key results

The potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50 ) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 μM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV 1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV 1.5.

Conclusion and implications

These results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation.

SUBMITTER: Burnham MP

PROVIDER: S-EPMC4294106 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Publications

Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

Burnham M P MP Sharpe P M PM Garner C C Hughes R R Pollard C E CE Bowes J J

British journal of pharmacology 20140813 21

<h4>Background and purpose</h4>Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV 1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling.<h4>Experimental approach</h4>Using the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for comp ...[more]

PMID: 24328991

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Project description:BackgroundDiabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM-with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated.Methods and resultsHere, we used the established streptozotocin-induced DCM murine model to investigate how hypoglycemia aggravates DCM progression. We confirmed connexin 43 (Cx43) dissociation from cell-cell interaction and accumulation at mitochondrial inner membrane both in the cardiomyocytes of patients with DM and DCM murine. Here, we observed that cardiac diastolic function, induced by chronic hyperglycemia, was further aggravated upon hypoglycemia challenge. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the mitogen-activated protein kinase kinase (MAPK kinase) (MEK)/extracellular regulated protein kinase (ERK) and inhibits phosphoinositide 3-kinase (PI3K)/Akt pathways, which results in Cx43 phosphorylation by Src protein and translocation to mitochondria in cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using adeno-associated virus serotype 2 (AAV2)/9. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiac diastolic dysfunction as well as aberrant electrophysiology in vivo. Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.ConclusionDCM presents compensatory adaptation of mild mtCx43 accumulation, yet acute hypoglycemia challenges result in further accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in hearts of patients with DM hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM.

Project description:Connexin 43 (Cx43) plays a crucial role in maintaining synchronous contraction in the heart. However, it remains unclear whether Cx43 directly influences the contractile force and synchrony of entire cardiac tissues. Previously, we successfully developed human-induced pluripotent stem cell (hiPSC)-derived cardiac tissues capable of directly measuring both the contractile force of the entire tissue and cellular synchrony within it. This study aimed to evaluate whether regulating GJA1, the gene encoding Cx43, could enhance contractility and synchrony in these tissues. Using adeno-associated virus (AAV), we mediated GJA1 overexpression (OE) or knockdown (shGJA1) in bioengineered hiPSC-derived cardiac tissues. Under electrical stimulation at 60 ppm, there were no significant differences in contractile force between the AAV-GJA1-OE and control tissues (0.78 ± 0.39 vs. 0.98 ± 0.43 mN, p = 0.32). Synchrony levels were also similar between these groups (p = 0.20). In contrast, shGJA1 tissues demonstrated significantly higher contractile force compared to scramble controls (1.55 ± 0.38 vs. 1.20 ± 0.15 mN, p = 0.039), although the difference in synchrony was not statistically significant (p = 0.08). RNA sequencing data revealed that a total of 37,199 genes were detected, comparing AAV6-GFP control and GJA1-OE treated hiPSC-CMs, as well as AAV6-shRNA scramble and shGJA1 treated hiPSC-CMs. We highlighted several candidate genes potentially contributing to the enhanced contractile force observed in the shGJA1 group. Furthermore, nineteen common genes were identified between the upregulation of shGJA1 compared to scramble and downregulation of GJA1-OE compared to control, which were associated with cell proliferation, transcription, contraction, and BMP signaling pathways. In conclusion, Cx43-OE did not appear to influence contractility and synchrony, meanwhile, Cx43 suppression may effectively improve contractility without impairing the synchrony in the entire cardiac tissues. Cx43 expression beyond a certain threshold may be sufficient to maintain synchronous contraction in the tissues.

Dataset Information

Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

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