Proceedings of the National Academy of Sciences, 2013
Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, re... more Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca 2+ -activated K + channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca 2+ -activated K + channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers.
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology... more Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the " voltage-clock, " where the hyperpolarization-activated funny current I f causes diastolic depolarization that triggers action potential cycling; and (ii) the " Ca 2+ clock, " where cyclical release of Ca 2+ from Ca 2+ stores depolarizes the membrane during diastole via activation of the Na + –Ca 2+ exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current-and voltage-clamp recordings from the same cell showed the so-called " voltage and Ca 2+ clock " pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the " voltage or Ca 2+ clock " produced a similar depo-larization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca 2+-activated intermediate K + conduc-tance (IK Ca , KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK Ca inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK Ca appears to play a crucial role in human embryonic cardiac automaticity. Ca2 +-activated K + channel SK4 | voltage clock | calcium clock | Na +-Ca 2+ exchanger | hyperpolarization-activated cyclic nucleotide-gated channel W hereas in early embryonic stages all cardiomyocytes are initially endowed with pacemaker activity, during heart development , most cardiac cells will differentiate into working myo-cardium lacking pacemaker properties. Only a small population of embryonic cardiomyocytes will form the sinoatrial node (SAN), the atrioventricular node, and the bundle of His (1). A crucial requirement for rhythmic automaticity is the existence of inward currents at diastolic potentials and a subtle dynamic integration of sarcolemmal ion channels, transporters, and Ca 2+ cycling proteins (2). Various ionic currents finely orchestrate rhythmic automaticity and are referred to as a voltage clock, including the pacemaker or funny current (I f), L-type, and T-type Ca 2+ currents (3–10). A Ca 2+-dependent pacemaker mechanism referred to as a Ca 2+ clock was also suggested to be a major player for automa-ticity, where the rhythmic local Ca 2+ release from the sarcoplasmic reticulum (SR) drives SAN pacemaker activity. SR Ca 2+ release via ryanodine receptors (RyRs) is thought to activate the forward mode of the electrogenic sarcolemmal Na + –Ca 2+ exchanger (NCX), which generates an inward current contributing to the late diastolic depolarization (DD), before the next action potential (AP) (7, 11). However, the cardiac pacemaker mechanisms remain unclear and controversial (12–15). Embryonic stem cells differentiate in vitro into spontaneously beating multicellular cardiomyocyte clusters within embryoid bodies (EBs), while recapitulating developmental stages of em-bryonic cardiomyogenesis (16–27). Thus, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) may provide insights into the pacemaker mechanisms of embryonic cardiac development. In this work, we used the current-and voltage-clamp configurations of the whole-cell patch-clamp technique to record, in the same beating hESC-CM, basal automaticity and ionic currents. This analysis revealed three main pacemaker phenotypes. The first was highly sensitive to I f current inhibition and insensitive to NCX blockade (28–30). The second cell population exhibited a pacemaker phenotype insensitive to I f current inhibition, but highly responsive to NCX blockers. The third hESC-CM population displayed pacemaker features that were sensitive to both I f and NCX blockers, indicating that voltage and Ca 2+ dependent pacemaker mechanisms can coexist in the same cell. Following exposure to blockers, all three pacemaker phenotypes shared a depolarizing drift of the maximal diastolic potential (MDP) that culminated by cessation of APs, suggesting that they converge to a common pacemaker component, which we identified by patch-clamp recording, real-time PCR, Western blotting, and immunocy-tochemistry as belonging to the intermediate-conductance Ca 2+-activated K + channels (IK Ca , KCa3.1 or SK4). Remarkably, IK Ca blockers (31, 32) inhibited the pacemaker in beating hESC-CMs, thereby leading to bradycardia, MDP depolarization, and ultimate suppression of automaticity. The pacemaker activity was sensitive Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca 2+-activated K + channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca 2+-activated K + channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers.
Proceedings of the National Academy of Sciences, 2013
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology... more Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.
Proceedings of the National Academy of Sciences, 2013
Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, re... more Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca 2+ -activated K + channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca 2+ -activated K + channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers.
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology... more Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the " voltage-clock, " where the hyperpolarization-activated funny current I f causes diastolic depolarization that triggers action potential cycling; and (ii) the " Ca 2+ clock, " where cyclical release of Ca 2+ from Ca 2+ stores depolarizes the membrane during diastole via activation of the Na + –Ca 2+ exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current-and voltage-clamp recordings from the same cell showed the so-called " voltage and Ca 2+ clock " pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the " voltage or Ca 2+ clock " produced a similar depo-larization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca 2+-activated intermediate K + conduc-tance (IK Ca , KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK Ca inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK Ca appears to play a crucial role in human embryonic cardiac automaticity. Ca2 +-activated K + channel SK4 | voltage clock | calcium clock | Na +-Ca 2+ exchanger | hyperpolarization-activated cyclic nucleotide-gated channel W hereas in early embryonic stages all cardiomyocytes are initially endowed with pacemaker activity, during heart development , most cardiac cells will differentiate into working myo-cardium lacking pacemaker properties. Only a small population of embryonic cardiomyocytes will form the sinoatrial node (SAN), the atrioventricular node, and the bundle of His (1). A crucial requirement for rhythmic automaticity is the existence of inward currents at diastolic potentials and a subtle dynamic integration of sarcolemmal ion channels, transporters, and Ca 2+ cycling proteins (2). Various ionic currents finely orchestrate rhythmic automaticity and are referred to as a voltage clock, including the pacemaker or funny current (I f), L-type, and T-type Ca 2+ currents (3–10). A Ca 2+-dependent pacemaker mechanism referred to as a Ca 2+ clock was also suggested to be a major player for automa-ticity, where the rhythmic local Ca 2+ release from the sarcoplasmic reticulum (SR) drives SAN pacemaker activity. SR Ca 2+ release via ryanodine receptors (RyRs) is thought to activate the forward mode of the electrogenic sarcolemmal Na + –Ca 2+ exchanger (NCX), which generates an inward current contributing to the late diastolic depolarization (DD), before the next action potential (AP) (7, 11). However, the cardiac pacemaker mechanisms remain unclear and controversial (12–15). Embryonic stem cells differentiate in vitro into spontaneously beating multicellular cardiomyocyte clusters within embryoid bodies (EBs), while recapitulating developmental stages of em-bryonic cardiomyogenesis (16–27). Thus, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) may provide insights into the pacemaker mechanisms of embryonic cardiac development. In this work, we used the current-and voltage-clamp configurations of the whole-cell patch-clamp technique to record, in the same beating hESC-CM, basal automaticity and ionic currents. This analysis revealed three main pacemaker phenotypes. The first was highly sensitive to I f current inhibition and insensitive to NCX blockade (28–30). The second cell population exhibited a pacemaker phenotype insensitive to I f current inhibition, but highly responsive to NCX blockers. The third hESC-CM population displayed pacemaker features that were sensitive to both I f and NCX blockers, indicating that voltage and Ca 2+ dependent pacemaker mechanisms can coexist in the same cell. Following exposure to blockers, all three pacemaker phenotypes shared a depolarizing drift of the maximal diastolic potential (MDP) that culminated by cessation of APs, suggesting that they converge to a common pacemaker component, which we identified by patch-clamp recording, real-time PCR, Western blotting, and immunocy-tochemistry as belonging to the intermediate-conductance Ca 2+-activated K + channels (IK Ca , KCa3.1 or SK4). Remarkably, IK Ca blockers (31, 32) inhibited the pacemaker in beating hESC-CMs, thereby leading to bradycardia, MDP depolarization, and ultimate suppression of automaticity. The pacemaker activity was sensitive Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca 2+-activated K + channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca 2+-activated K + channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers.
Proceedings of the National Academy of Sciences, 2013
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology... more Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.
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