Cardiac-specific overexpression of Gαq alters excitation–contraction coupling in isolated cardiac myocytes

doi:10.1006/jmcc.1999.0966

Journal of Molecular and Cellular Cardiology

Volume 31, Issue 7, July 1999, Pages 1327-1336

https://doi.org/10.1006/jmcc.1999.0966 Get rights and content

Abstract

Transgenic mice with cardiac-specific overexpression of Gαq exhibit a biochemical and physiological phenotype of load-independent cardiac hypertrophy with contractile dysfunction. To elucidate the cellular basis for altered contractility, we measured cellular contraction, Ca²⁺transients, andl-type Ca²⁺channel currents (I_Ca) in left ventricular (LV) myocytes isolated from non transgenic (NT) controls or Gαq hearts. Although baseline contractile function (% shortening) and the amplitude of Ca²⁺transients in Gαq myocytes were similar to NT myocytes, the rates of cellular shortening and relengthening and the duration of Ca²⁺transients were prolonged in Gαq myocytes. Myocytes from Gαq hearts had larger cell capacitance but no change in I_Cadensity, voltage-dependence of activation and inactivation. The responses of I_Cato dihydropyridine drugs and a membrane permeable cAMP analog, 8-(4-chlorophenylthio) cAMP, were not altered; however, the time course of I_Cainactivation was significantly slower in Gαq myocytes compared to NT myocytes. The kinetic difference in inactivation was abolished when Ba²⁺was used as the charge carrier or when the sarcoplasmic reticulum (SR) Ca²⁺was depleted by ryanodine, suggesting that Ca²⁺-dependent inactivation is reduced in Gαq myocytes due to altered SR Ca²⁺release. Consistent with this hypothesis, the function of SR as assessed by the maximal Ca²⁺uptake rates and the apparent affinity of SR Ca²⁺-ATPase for Ca²⁺was reduced in ventricles of Gαq heart. These results suggest that the reduced SR function contributes to the depressed contractility associated with this form of cardiac hypertrophy.

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Cited by (35)

The atypical 'b' splice variant of phospholipase Cβ1 promotes cardiac contractile dysfunction
2015, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
Conditional overexpression in adult hearts of GαqQ209L, like Gαq-WT, does not result in hypertrophy, but instead initiates a rapid loss of contractility, along with chamber dilatation and subsequent heart failure [43]. The reduced contractility, associated with depressed SR Ca2 + cycling [44] is reminiscent of responses to PLCβ1b, although the phenotype is more complex and more severe following conditional expression of GαqQ029L. Significantly, all of these responses to GαqQ209L appeared to require PLCβ activation, because a GαqQ209L mutant unable to activate PLCβ subtypes (GαqQ209LAA) did not cause any of these changes.
The activity of the early signaling enzyme, phospholipase Cβ1b (PLCβ1b), is selectively elevated in diseased myocardium and activity increases with disease progression. We aimed to establish the contribution of heightened PLCβ1b activity to cardiac pathology. PLCβ1b, the alternative splice variant, PLCβ1a, and a blank virus were expressed in mouse hearts using adeno-associated viral vectors (rAAV6-FLAG-PLCβ1b, rAAV6-FLAG-PLCβ1a, or rAAV6-blank) delivered intravenously (IV). Following viral delivery, FLAG-PLCβ1b was expressed in all of the chambers of the mouse heart and was localized to the sarcolemma. Heightened PLCβ1b expression caused a rapid loss of contractility, 4–6 weeks, that was fully reversed, within 5 days, by inhibition of protein kinase Cα (PKCα). PLCβ1a did not localize to the sarcolemma and did not affect contractile function. Expression of PLCβ1b, but not PLCβ1a, caused downstream dephosphorylation of phospholamban and depletion of the Ca^2 + stores of the sarcoplasmic reticulum. We conclude that heightened PLCβ1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCβ1b is a cardiac-specific signaling system, and thus provides a potential therapeutic target for the development of well-tolerated inotropic agents for use in failing myocardium.
Cardiac-directed expression of adenylyl cyclase reverses electrical remodeling in cardiomyopathy
2006, Journal of Molecular and Cellular Cardiology
Adenylyl cyclase (AC) is an effector molecule in β-adrenergic receptor signaling, catalyzing conversion of ATP to cAMP. Agents that increase intracellular levels of cAMP have been used previously to treat clinical heart failure. Recently, Roth et al. have shown that long-term cardiac-directed expression of AC_VI, a dominant AC isoform in mammalian cardiac myocytes, increases survival and abrogates myocardial hypertrophy in transgenic (TG) mice with G_αq-associated cardiomyopathy. Indeed, it has been proposed that increasing the cardiac content of AC_VI is fundamentally different than other strategies used to increase cAMP function. However, one important but unexplored issue is its effects on electrical remodeling. Electrophysiological properties of G_αq mice have been characterized. Similar to other models of cardiac hypertrophy and failure, cardiac myocytes isolated from G_αq mice show prolonged action potential with reduced transient outward K⁺ current (I_to) and inward rectifier K⁺ current (I_K1) density compare to wild-type (WT) animals. We directly examined the electrical remodeling of cardiac-directed AC_VI over-expression in G_αq mice using ECG recordings and whole-cell patch-clamp recordings. Four groups of animals were used: WT (double negative), AC_VI, G_αq and double positive TG mice (G_αq/AC_VI). Cardiac-directed expression of AC_VI results in the reversal of adverse electrical remodeling in the G_αq mice and is associated with significant improvement in the delay of cardiac repolarization and arrhythmias. Specifically, there is a normalization of I_to, I_K1 and action potential duration in G_αq/AC_VI compared to G_αq mice. In summary, our data provide evidence that increased cardiac AC_VI content has a salutary effect in cardiomyopathy and cardiac electrical remodeling.
Down regulation of the L-type Ca<sup>2+</sup> channel, GRK2, and phosphorylated phospholamban: protective mechanisms for the denervated failing heart
2006, Journal of Molecular and Cellular Cardiology
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Myocardial cells were filtered through a 200 μm nylon mesh and stored in Krebs–Henseleit solution containing 1 mmol/l Ca2+. Myocyte contraction and relaxation functions were measured using a video motion edge detector at 32 °C, as previously described in [30,31]. Whole-cell ICa were characterized using patch-clamp techniques at room temperature (22–23 °C), as previously described in [32,33].
We previously found that a canine model of selective surgical ventricular denervation (VD), which does not permit increased sympathetic tone during the pathogenesis of heart failure (HF), tolerated the development of HF better than controls. To investigate the cellular mechanisms, we examined cellular contraction and L-type Ca²⁺ channel currents (I_Ca) and their responses to β-adrenergic receptor (β-AR) stimulation in left ventricular myocytes from 1) control, 2) VD, 3) HF induced by rapid pacing, and 4) HF induced in VD (VD-HF) dogs. The magnitude of myocyte contraction and rate of relaxation in VD were similar to control but were depressed in both HF and VD-HF. These changes were associated with reduced protein expression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and protein kinase A phosphorylated phospholamban (PLB), which was reduced in HF, but essentially abolished in VD-HF. β-AR kinase (GRK2) was increased in HF but reduced in VD-HF. Basal I_Ca density did not differ among control, VD, and HF groups, but VD-HF myocytes showed a markedly reduced I_Ca density (~40%). Compared to controls, the sensitivity of I_Ca to isoproterenol (ISO), was significantly higher in VD, but reduced in HF. While I_Ca responses to ISO in VD-HF were maintained at control levels, the amplitude of the ISO-stimulated I_Ca was significantly smaller (~50%) compared with HF myocytes. The relative decrease in Ca²⁺ influx due to downregulation of I_Ca density may contribute to the cardioprotective effects in VD-HF hearts by preventing Ca²⁺ overload during the development of HF. These findings, in combination with the virtual abolition of phosphorylated PLB in VD-HF and the decrease in GRK2, may explain, in part, why VD dogs tolerate the development of HF better than control dogs.
Mechanism of Enhanced Cardiac Function in Mice with Hypertrophy Induced by Overexpressed Akt
2003, Journal of Biological Chemistry
Citation Excerpt :
The inactivation kinetics of ICa at +10 mV was significantly faster in TG myocytes compared with WT myocytes (Fig. 2B). In mouse ventricular myocytes, ICa inactivation is controlled by Ca2+ in the sarcolemmal subspace (19, 23, 24). To examine whether the difference in ICa inactivation rate between WT and TG myocytes was due to the contribution of Ca2+ released from the SR, the SR Ca2+ content was depleted by ryanodine (10 μm).
Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3–4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 ± 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 ± 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 ± 0.2 versus 3.5 ± 0.2%, p < 0.01) was associated with increased Fura-2 Ca²⁺ transients (396 ± 50 versus 250 ± 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 ± 15 versus 98 ± 18 μm/s, p < 0.01). L-type Ca²⁺ current (I_Ca) density was increased in TG compared with WT (-9.0 ± 0.3 versus 7.2 ± 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca²⁺ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca²⁺ transients, and Ca²⁺ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.
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This review briefly highlights the major mechanisms of left ventricular (LV) remodeling following myocardial infarction in heart failure and presents novel strategies to limit infarct size and retard the progression of LV remodeling. This is important since interventions that have been shown to improve mortality in patients with heart failure such as angiotensin converting enzyme inhibitor and β-blockers had a favorable impact on the LV remodeling process. Although several other animal models of heart failure are available, we chose the rat infarct model because of its accurate reflection of human pathophysiology. It is clear from this review that the understanding of the mechanisms responsible for the regression of ventricular remodeling is still unclear. Further investigations in this area will certainly lead to the development of new therapy for heart failure.
Physiological determinants of contractile force generation and calcium handling in mouse myocardium
2002, Journal of Molecular and Cellular Cardiology
L. B. Stull, M. K. Leppo, E. Marbán and P. M. L. Janssen. Physiological Determinants of Contractile Force Generation and Calcium Handling in Mouse Myocardium. Journal of Molecular and Cellular Cardiology (2002) 34, 1367–1376. Despite the fact that the mouse has become a common tool to study cardiac dysfunction, little is known regarding the regulation of murine cardiac contractility. We have investigated the three main mechanisms that regulate cardiac output (frequency-dependent activation, length-dependent activation, and β-adrenergic stimulation) in ultra-thin right ventricular (RV) trabeculae from the mouse heart at body temperature (37°C). [Ca²⁺]_i was recorded in a subset of trabeculae iontophoretically loaded with fura-2, and rapid cooling contractures were performed to estimate the sarcoplasmic reticulum (SR) calcium load. The force–frequency relationship was positive (2–12 Hz); force increased, albeit slightly, while relaxation timing decreased. As expected, in response to β-adrenergic stimulation, force development increased while contractile duration decreased, and increased muscle length led to increased force generation. Changes in SR calcium load and the calcium transient amplitude paralleled effects on active force generation. Despite several qualitative similarities with other mammalian species, the reserve for augmentation of force via either increased frequency or β-adrenergic stimulation was considerably smaller in mouse than in other animals. Therefore, changes in preload, as opposed to increased HR or adrenergic tone, appears to be a much more important determinant of cardiac performance in the mouse than in larger mammals.

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^f1: Please address all correspondence to: Dr Atsuko Yatani, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575. E-mail: [email protected]

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Journal of Molecular and Cellular Cardiology

Feature ArticleCardiac-specific overexpression of Gαq alters excitation–contraction coupling in isolated cardiac myocytes

Abstract

The atypical 'b' splice variant of phospholipase Cβ1 promotes cardiac contractile dysfunction

Cardiac-directed expression of adenylyl cyclase reverses electrical remodeling in cardiomyopathy

Down regulation of the L-type Ca<sup>2+</sup> channel, GRK2, and phosphorylated phospholamban: protective mechanisms for the denervated failing heart

Mechanism of Enhanced Cardiac Function in Mice with Hypertrophy Induced by Overexpressed Akt

Ventricular remodeling in heart failure

Physiological determinants of contractile force generation and calcium handling in mouse myocardium

Feature Article
Cardiac-specific overexpression of Gαq alters excitation–contraction coupling in isolated cardiac myocytes