Restoration of defective L-type Ca²⁺ current in cardiac myocytes of type 2 diabetic db/db mice by Akt and PKC-Ι

Abstract

Diabetes is associated with increased risk of heart failure and development of a cardiomyopathy whose etiology is only partially understood. Ca²⁺ entry through the voltage-dependent L-type Ca²⁺ channel Ca_V1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed Ca_V1.2 function have been reported in obese type 2 diabetic db/db mice. Here we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of Ca_V1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca²⁺ current (I_Ca,L) density nearly to the level seen in wildtype cells. PIP3 also reversed the positive shift in the voltage dependence of steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected I_Ca,L. Akt1 and Akt2 were as effective as PIP3 in restoring I_Ca,L density in db/db myocytes, but did not affect the voltage dependence of current activation. Infusion of atypical PKC-Ι (the human homolog of mouse PKC-λ) caused a small but significant increase in I_Ca,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed Ca_V1.2 function in the heart of type 2 diabetic db/db mice.

INTRODUCTION

Cardiovascular complications such as coronary artery disease, stroke, and peripheral arterial disease are responsible for most of the morbidity and mortality suffered by diabetic patients.¹ In addition, diabetic patients have a greater risk of developing heart failure than non-diabetic patients, even after controlling for factors such as coronary artery disease and hypertension. The underlying pathogenesis of this diabetic cardiomyopathy is only partially understood, and is thought to include changes in myocardial structure, metabolism and Ca²⁺ cycling.^2,3 The voltage-dependent L-type Ca²⁺ channel Ca_V1.2 is the critical initiator of the contractile cycle in ventricular cardiac myocytes, and inhibition of Ca_V1.2 function diminishes Ca²⁺ entry and contractile force. Studies have shown that Ca²⁺ current through Ca_V1.2 (I_Ca,L) is reduced in cardiac myocytes from type 1 diabetic Ins2^Akita and type 2 diabetic db/db mice.^{4, 5} Cardiac contractility in both types of diabetic mice has also been shown to be impaired.^{4, 5}

Many of the metabolic effects of insulin are thought to be mediated by phosphoinositide 3-kinase (PI3K) signaling. PI3Ks are activated in response to insulin or other growth factors and phosphorylate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) to form the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3)⁶. As a consequence of increased PIP3 production, downstream effectors including members of the protein kinase Akt family (also known as PKB) and atypical PKC-Ι/λ become phosphorylated and activated.^{7, 8} Experiments in knockout mice have shown that both Akt2 and PKC-λ (the mouse homolog of human PKC-Ι) contribute to insulin-stimulated glucose uptake in the heart.^{9, 10} The PI3K signaling pathway also modulates cardiac contractile function.¹¹ Ablation of the p110γ PI3K isoform in mice resulted in enhanced cardiac contractility due to an increase in cAMP levels.¹² By contrast, we found that mice lacking the p110α PI3K isoform in the heart exhibited decreased cardiac contractility in vivo and reduced I_Ca,L in isolated myocytes.¹³ We used a number of methods to manipulate PI3K/PIP3/Akt signaling to demonstrate that activation of this pathway is needed to maintain normal I_Ca,L and myocyte contractility.^{13, 14} Other investigators have shown that transgenic mice overexpressing constitutively active forms of p110α or Akt in cardiac myocytes exhibited enhanced cardiac contractility and increases in Ca_V1.2 expression or I_Ca,L.^{15, 16} To our knowledge, the effect of PKC-Ι/λ on cardiac contractility or I_Ca,L has not been examined.

Reduced insulin activation of PI3K signaling due to either lack of insulin (type 1) or insulin resistance (type 2) is a well-studied aspect of diabetes, especially in the context of glucose uptake. In a previous study, we determined that decreased I_Ca,L in cardiac myocytes from non-obese type 1 diabetic Ins2^Akita mice is due mainly to a reduction in PI3K activity.⁵ These results were surprising, considering the long-term hyperglycemia, multiple metabolic disturbances and other physiological changes in these mice that could have affected channel function. This led us to speculate that low PI3K signaling is also responsible for the reduction of I_Ca,L in cardiac myocytes from obese type 2 diabetic db/db mice⁴. In this study, we show that decreased I_Ca,L in cardiac myocytes from db/db mice is due in large part to decreased PI3K signaling. In addition, we demonstrate that Akt and PKC-Ι differentially regulate Ca_V1.2 function.

MATERIALS & METHODS

Materials

Recombinant human PKC-Ι was from Invitrogen (Carlsbad, CA, USA). The active Akt1, active Akt2 and unactive Akt1 were from Millipore (Billerica, MA, USA). Phosphoinositides (all di-C8) were from Echelon Biosciences (Salt Lake City, UT, USA). Ventricular myocyte isolation—Male db/db (B6.BKS(D)-Lepr^db/J) and WT control (C57BL/6J) mice were purchased from Jackson Laboratories and used at 4-5 months of age. Hyperglycemic status of the db/db animals was confirmed by measuring tail vein glucose levels using a glucometer (OneTouch UltraMini, LifeScan, Inc., Milpitas, CA, USA). Mice were euthanized by intraperitoneal injection of 100 mg sodium pentobarbital per kg of body weight and ventricular myocytes were isolated as previously described¹⁴. All animal-related experimental protocols were approved by the Institutional Animal Care and Use Committee of Stony Brook University.

Electrophysiology

Whole-cell patch clamping of isolated cardiac myocytes was performed as previously described.¹⁴ In brief, only clearly rod-shaped myocytes were studied. Whole-cell patch clamp recordings used 2–3 M borosilicate glass pipettes measured prior to sealing (Sutter Instrument), pCLAMP 8 software, the DigiData 1350 interface, and the Axopatch 1D amplifier (Axon Instruments). For the recording of I_Ca,L, pipettes contained an internal solution (111 mM CsCl, 20 mM tetraethylammonium chloride, 10 mM glucose, 14 mM EGTA, 10 mM HEPES, and 5 mM MgATP, pH 7.2, adjusted with CsOH) and cells were perfused in a Na⁺-free bath solution (137 mM tetraethylammonium chloride, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.4, adjusted with tetraethylammonium hydroxide).

Data Analysis

Student’s t tests were performed to evaluate the significance of the differences between mean values. A value of P < 0.05 was considered significant.

RESULTS

PIP3 Increases I_Ca,L Density in Diabetic db/db Myocytes

Diabetic db/db mice exhibit a cardiac contractility defect and reduced I_Ca,L density in myocytes⁴. We confirmed that I_Ca,L density is reduced in db/db cardiac myocytes as compared to WT cells. Fig. 1A (upper panels) shows typical recordings of I_Ca,L activation in myocytes isolated from WT and diabetic db/db mice. I_Ca,L was elicited by depolarizing voltage pulses from −50 mV to +50 mV in 10 mV increments (300 ms duration) from a holding potential of −50 mV. The membrane capacitance was measured by applying a hyperpolarizing voltage step from a holding potential of −50 mV, and the current amplitudes were normalized to cell capacitance to obtain current density in pA/pF. Membrane capacitance, which is proportional to surface area, was not significantly different between the two groups of myocytes (148.0 ± 9.8 pF in WT (n = 31) vs. 145.0 ± 8.5 pF in db/db (n = 45)). The peak values from these recordings were used to construct current density-voltage (I-V) relationships that show a substantial reduction in I_Ca,L density in db/db myocytes as compared to WT cells at most of the voltages tested (lower panel of Fig. 1A). To determine if decreased PI3K signaling in diabetic hearts might be responsible for this difference, we infused the cells with PIP3 through the patch pipette. PIP3 had little effect on WT cells but significantly enhanced I_Ca,L density in the db/db myocytes at most of the voltages tested (Fig. 1B).

Figure 1. PIP3 increases I_Ca,L density in myocytes from db/db mice.

(A) I_Ca,L activation in myocytes from WT or db/db mice infused with internal solution. Upper panels: Representative recordings of I_Ca,L activation. Lower panel: The corresponding peak current density-voltage (I-V) relationships of I_Ca,L activation. (B) I_Ca,L activation in myocytes from WT or db/db mice infused with internal solution containing 1 μM PIP3. Upper panels: Representative recordings of I_Ca,L activation. Lower panel: The corresponding I-V relationships. n = 10 cells for each group.

We performed additional experiments using control phosphoinositides to determine if the stimulatory effect was specific to PIP3. I_Ca,L density was measured at +10 mV following a single depolarizing step of 300 ms duration from a holding potential of −50 mV. The peak I_Ca,L density at +10 mV in db/db myocytes in the absence of PIP3 was 53% lower than in WT cells (Fig. 2). Infusion of PIP3 increased I_Ca,L density in the db/db cells to nearly the wildtype level, but the control phosphoinositides PI(3,5)P₂ or PI(4,5)P₂ did not have a stimulatory effect (Fig. 2). These results are consistent with the hypothesis that decreased I_Ca,L density in db/db diabetic myocytes is due mostly to reduced PI3K activity.

Figure 2. Effect of PI3K effectors and isoproterenol (ISO) on I_Ca,L density.

Summarized I_Ca,L density at +10 mV in myocytes from WT and db/db mice. Phosphoinositides (1 μM) and kinases (20 nM) in internal solution were infused through the patch pipette and ISO (5 μM) was applied to the external solution. ** indicates a significant difference vs. db/db control (p<0.01). The number of cells studied in each group is above each bar.

Next we examined the time-dependent effect of PIP3 on I_Ca,L activation. In this protocol, the cells were repeatedly depolarized every 15 s with voltage steps 300 ms in duration to +10 mV from a holding potential of −50 mV. The typical “run-down” of I_Ca,L density was observed in both WT and diabetic myocytes under the control conditions, and current density decreased by 20% and 24%, respectively, within 240 s (Fig. 3A). Infusion of WT myocytes with PIP3 prevented the “run-down” and caused I_Ca,L density to increase slightly over time (left panel of Fig. 3A). A much larger “run-up” was seen in db/db myocytes in the presence of PIP3, and a plateau was reached after about 150 s (right panel of Fig. 3A). Plateau values for I_Ca,L density in the presence of PIP3 were approximately 11 pA/pF (WT) and 9 pA/pF (db/db). These results indicate that I_Ca,L density in diabetic myocytes is increased to its maximum value within minutes after PI3K signaling is artificially enhanced by intracellular infusion of PIP3.

Figure 3. Time-dependent stimulation of I_Ca,L by PIP3 and ISO.

Time dependence of I_Ca,L activation in myocytes from WT (left panels) and db/db (right panels) mice infused with or without 1 μM PIP3 (A), treated with extracellular application of 5 μM ISO (B), or infused with PIP3 and then treated with ISO (C). n = 10 cells for each group.

Independent Effects of Isoproterenol and PIP3 on I_Ca,L Stimulation in db/db Myocytes

β-adrenergic stimulation of I_Ca,L is a well-known mechanism that regulates cardiac contraction. To determine if a defect in β-adrenergic regulation is responsible for the suppression of I_Ca,L in db/db myocytes, we treated cells with the β-adrenergic agonist isoproterenol (ISO). ISO stimulated I_Ca,L in a time-dependent manner in both WT and db/db myocytes (Fig. 3B). Infusion of WT myocytes with PIP3 caused a small increase in I_Ca,L density that was further enhanced to a much larger extent by subsequent application of ISO (left panel of Fig. 3C). PIP3 caused a comparatively larger increase in current density in diabetic cells that was also further increased in the presence of ISO (right panel of Fig. 3C). Summary data for current density at +10 mV show that ISO increased I_Ca,L by 1.4-fold in both WT and db/db cells (Fig. 2). However, total current density in the presence of ISO plus PIP3 was much lower in db/db cells than in WT myocytes (Fig. 2). These results indicate that β-adrenergic regulation of channel function is intact in the diabetic cells and suggest that modulation of I_Ca,L by PIP3 is independent of PKA.

Akt and PKC-Ι Increase I_Ca,L Density in db/db Myocytes

The protein kinases Akt1, Akt2 and PKC-λ/Ι are activated downstream of PI3K. We next asked if either Akt or PKC-λ/Ι would mimic the stimulatory effect of PIP3 on I_Ca,L density in diabetic myocytes. Indeed, infusion of db/db myocytes with Akt1 or Akt2 caused a significant enhancement of I_Ca,L density at most of the voltages tested, whereas unactivated Akt1 had little or no effect (Fig. 4). Because mouse PKC-λ is not commercially available, we used the human homolog PKC-Ι in infusion experiments. PKC-Ι also caused an increase in I_Ca,L density at most of the voltages tested (Fig. 4). Summary data for current density at +10 mV show that infusion of db/db myocytes with either Akt1 or Akt2 enhanced I_Ca,L density to nearly the level in WT cells, and the effects were comparable to those obtained with PIP3 (Fig. 2). Although the increase in I_Ca,L with PKC-Ι was significant (P<0.05 as compared to db/db control), the average I_Ca,L density was less than that seen with PIP3, Akt1 or Akt2 (Fig. 2). Thus, Akt and, to a lesser extent, PKC-Ι, partially reversed the defect in I_Ca,L density in myocytes from db/db mice.

Figure 4. Akt and PKC-Ι increase I_Ca,L density in db/db myocytes.

Upper panels: Representative recordings of I_Ca,L activation in db/db myocytes infused with internal solution without (control) or with inactive Akt1 or active Akt1, Akt2 or PKC-Ι (20 nM each). Lower panel: The corresponding I-V relationships for I_Ca,L activation. The voltage protocol was the same as for Fig. 1.

Effect of PIP3 on Steady-state Activation, Inactivation and Recovery Kinetics of I_Ca,L

The decrease in I_Ca,L density in db/db diabetic myocytes has been shown to be due in part to altered voltage dependence that affects the steady-state activation of I_Ca,L.⁴ To determine the effect of PIP3 on steady-state activation, cells were exposed to depolarizing voltage steps from −50 mV to +10 mV (300 ms duration) from a holding potential of −50 mV and the data were fit to a Boltzmann 2-state model. We confirmed the earlier report⁴ that the steady-state activation curve for db/db myocytes was shifted toward more positive potentials as compared to the WT curve (Fig. 5A) and there was a significant difference between the voltages at 50% activation (V_h) for WT and db/db cells (Table 1). Infusion of WT cells with PIP3 did not alter the steady state activation of I_Ca,L, whereas infusion of db/db cells caused the curve to shift to the left so that it was nearly superimposed on the WT curves (Fig. 5A). V_h was essentially the same for WT cells (plus or minus PIP3) and db/db cells in the presence of PIP3 (Table 1). There were no differences in the corresponding slope factors (K) among the four groups (P>0.05 when compared to WT control) (Table 1). These results indicate that the change in steady-state activation of I_Ca,L in db/db myocytes is completely reversed by PIP3.

Figure 5. Effect of PI3K effectors on steady-state activation of I_Ca,L.

(A) Steady-state activation curves of I_Ca,L in WT and db/db myocytes infused with or without 1 μM PIP3. (B) Steady-state activation curves of ICa,L in db/db myocytes infused with inactive Akt1 or active Akt1, Akt2 or PKC-Ι (20 nM each). Biophysical parameters derived from each curve and the number of cells in each group are shown in Table 1.

Table 1.

Biophysical properties of I_Ca,L in WT and db/db myocytes

		Steady-State Activation		Steady-State Inactivation		Recovery from Inactivation τ (ms)
		Vh (mV)	K (mV)	V1/2 (mV)	K (mV)
WT	Control	−17.8 ± 0.7 (10)	3.3 ± 0.6 (10)	−30.4 ± 1.1 (10)	4.6 ± 1.1 (10)	330 (7)
	PIP3	−17.9 ± 0.8 (7)	3.4 ± 0.4 (7)	−31.0 ± 1.2 (5)	4.5 ± 1.0 (5)	328 (5)
db/db	Control	−13.8 ± 0.4 (10)**	5.0 ± 0.3 (10)	−28.9 ± 1.0 (10)	4.8 ± 0.9 (10)	365 (5)
	PIP3	−18.5 ± 0.6 (10)##	4.0 ± 0.6 (10)	−28.9 ± 1.6 (10)	4.0 ± 1.4 (10)	348 (5)
	Unact Akt1	−14.0 ± 0.8 (7)	4.6 ± 0.6 (7)
	Act Akt1	−15.6 ± 0.3 (10)	4.5 ± 0.2 (10)
	Act Akt2	−15.2 ± 0.6 (10)	4.0 ± 0.4 (10)
	PKC-I	−18.7 ± 0.6 (7)##	4.3 ± 0.4 (7)

Numbers in parentheses indicate the number of myocytes tested.

^**significantly different from WT control (P < 0.01).

^##significantly different from db/db control (P < 0.01).

To investigate the steady-state inactivation of I_Ca,L, cells were subjected to conditioning pulses 2 s in duration from −80 mV to +40 mV in 10 mV increments from a holding potential of −50 mV followed by a test step to +10 mV. The data fit by a Boltzmann 2-state model showed that there was no significant difference in the membrane potential at which 50% of the channels are inactivated (V_1/2) or K between WT and db/db cells in the presence or absence of PIP3 (Table 1) (P>0.05 for all groups when compared to WT control, t-test). We then examined the effect of PIP3 on the kinetics of recovery from inactivation of I_Ca,L using a 2-pulse protocol. First, a depolarizing pulse 300 ms in duration was applied from a holding potential of −50 mV to the test potential of +10 mV to activate I_Ca,L. A variable interval was allowed for recovery, then a second identical test pulse was applied. The cycle length was 8 s. The data were fit to a single exponential using the equation I/I_max=1-e^(−t/τ) to calculate the average time constant (τ) of I_Ca,L recovery at different test voltages. There were no significant differences in τ between WT and db/db myocytes in the presence or absence of PIP3 (P>0.05 for all groups when compared to WT control) (Table 1). These results indicate that the steady-state inactivation and recovery from inactivation of I_Ca,L are not altered in db/db myocytes.

PKC-Ι Modulates the Steady-State Activation of I_Ca,L in db/db Myocytes

We also examined the effect of Akt1, Akt2 and PKC-Ι on the steady-state activation of I_Ca,L. The steady-state activation curves generated in db/db cells infused with Akt1, Akt2 or inactive Akt1 were very similar to each other (Fig. 5B) and the V_h and K values were not significantly different as compared to the db/db control (Table 1) (P>0.05). In contrast, PKC-Ι infusion of db/db myocytes caused a negative shift of the steady-state activation curve (Fig. 5B), yielding V_h and K values that were statistically indistinguishable from those for control WT cells (P>0.05) (Table 1). In summary, PKC-Ι completely reversed the alteration in steady-state activation of I_Ca,L in db/db myocytes.

DISCUSSION

The number of diabetic adults worldwide in 2010 is estimated to be 285 million, and this number is expected to increase to 439 million by 2030.¹⁷ The total number of excess deaths in adults attributable to diabetes is estimated to be 3.96 million for the year 2010, accounting for 6.8% of all mortality.¹⁸ Approximately 90% of diabetic patients have type 2 diabetes. Since cardiovascular diseases are the most common cause of death in people with diabetes,¹ a better understanding of the mechanisms that cause cardiovascular complications in type 2 diabetes is urgently needed.

The db/db mouse is a well-studied model of obesity and type 2 diabetes. A previous study showed that decreased cardiac contractility in type 2 diabetic db/db mice is due to a defect in Ca²⁺ transients caused at least in part by a reduction in I_Ca,L density. A positive shift in the voltage dependence of steady-state activation of I_Ca,L was also noted.⁴ Here we show that these defects in I_Ca,L are mainly the result of low PI3K signaling. Infusion of diabetic cardiac myocytes with PIP3 or Akt increased I_Ca,L nearly to the level seen in control cells, and intracellular delivery of PKC-Ι corrected the alteration in voltage dependence of activation.

The relatively rapid and large increase in I_Ca,L density upon infusion of PIP3 or protein kinases that are activated upon production of PIP3 suggests that the db/db cells contain a pool of inactive channels that responds to an increase in PI3K signaling. We showed earlier that I_Ca,L in myocytes from type 1 diabetic Ins2^Akita mice is low because of decreased expression of Ca_V1.2 on the cell surface, and that PIP3 increases I_Ca,L density in these cells by promoting trafficking of the channel to the cell surface.⁵ L-type Ca²⁺ channels in the heart are composed of the pore-forming Ca_V1.2 subunit, which determines the principal biophysical and pharmacological properties of the channel, in a complex with accessory β₂ and α₂δ subunits that are thought to play a role in trafficking.^{19, 20} Akt phosphorylation of the β_2a subunit in transfected cells has been shown to increase Ca 2.2 N-type Ca²⁺ V channel activity by promoting trafficking of the channel to the plasma membrane.²¹ We assume that a similar Akt- (or, to a lesser extent, PKC-λ-) dependent trafficking mechanism is mainly responsible for the stimulatory effect of PI3K signaling on Ca_V1.2 function in the db/db diabetic cells.

Although infusion of db/db cells with PIP3 or Akt caused a significant increase in I_Ca,L density, the maximal current density we measured in the presence of PIP3 plus ISO was 37% lower in db/db cells than in wildtype cells. These results are consistent with a decrease in total expression of the Ca_V1.2 protein, as demonstrated by Pereira et al.⁵ Loss of Ca_V1.2 protein in the diabetic cells may also be due to suppressed PI3K signaling, as Akt-dependent phosphorylation of β₂ has been proposed to stabilize the Ca_V1.2 protein in cardiac myocytes. ²²

A comparison of the data herein with our earlier results using myocytes from Ins2^Akita type 1 diabetic mice⁵ indicates that there are some differences in the way I_Ca,L is affected in these two models of diabetes. First, the peak I_Ca,L density at +10 mV was reduced much more in db/db myocytes (53%) than in Ins2^Akita myocytes (38%) as compared to nondiabetic cells. Second, PIP3 partially restored I_Ca,L density in db/db myocytes but fully restored I_Ca,L density in Ins2^Akita myocytes. Both of these disparities are probably due to the reduced expression of Ca_V1.2 protein in db/db myocytes⁴ that is not seen in Ins2^Akita myocytes.⁵ These differences suggest that hyperglycemia in combination with obesity and insulin resistance in type 2 diabetes is more damaging to Ca_V1.2 function than hyperglycemia and lack of insulin in type 1 diabetes. Finally, myocytes from Ins2^Akita mice exhibited positive shifts in the voltage dependence of steady-state activation and inactivation of I_Ca,L and PIP3 infusion corrected the change in inactivation only, whereas PIP3 corrected the change in voltage dependence of steady-state activation in db/db cells.

Many ion channels, including Ca_V1.2, respond to depletion and resynthesis of phosphoinositides, especially PI(4,5)P₂. Alterations in channel activity and voltage dependence have been proposed to be mediated by binding of PI(4,5)P₂ to the channel proteins.^23-25 Our demonstration that intracellular delivery of Akt or PKC-Ι mimics the effects of PIP3 in db/db myocytes suggests that PIP3 alters Ca_V1.2 function indirectly via a phosphorylation mechanism. This idea is supported by our earlier study showing that I_Ca,L in myocytes lacking the p110α isoform of PI3K is increased by infusion of Akt1, and I_Ca,L in wildtype myocytes is decreased upon treatment with an Akt inhibitor.¹³ Phosphorylation is already known to be an important mechanism that regulates cardiac Ca_V1.2 function.²⁶ β-adrenergic stimulation of I_Ca,L in the heart is mediated by PKA-dependent phosphorylation of Ca_V1.2.²⁷ Phosphorylation of Ca_V1.2 by PKG and a number of PKC isoforms is also associated with changes in channel activity.^{28, 29} In addition, as discussed above, Akt phosphorylation of β₂ subunits promotes increased trafficking of the channel to the plasma membrane and stabilizes the Ca_V1.2 protein.^{21, 22} Thus, it is possible that downregulation of Ca_V1.2 function in db/db myocytes might involve reduced phosphorylation of Ca_V1.2 by PKC-λ that alters gating and reduced phosphorylation of β₂ by Akt and/or PKC-λ that decreases the total number of channels and the number of channels on the cell surface.

PI3K signaling pathways can be activated by a variety of hormones and may utilise distinct molecular cassettes to differentially regulate cardiac physiology. For example, cardiac contractility is positively regulated by the PI3K p110α isoform and negatively regulated by PI3K p110γ, whereas PI3K p110β promotes autophagy. ^{12, 13, 15, 30} In addition, Akt1 regulates heart size whereas Akt2 regulates cardiomyocyte metabolism and survival. ^{9, 31} Coupling of different PI3K signaling pathways to distinct responses may involve compartmentation of receptors, PI3K isoforms and downstream signaling molecules into signalosomes.³² Therefore, strategies that aim to increase overall PI3K signaling in diabetic patients might lead to detrimental effects on the heart and other organs. On the other hand, drugs that improve insulin sensitivity in the heart might provide protection from some of the deleterious effects of diabetes on cardiac function.

CONCLUSIONS

We conclude that reduced activation of the PI3K/Akt/PKC-λ signaling pathway in the heart of type 2 diabetic db/db mice causes a decrease in Ca_V1.2 function in these animals. We speculate that a similar defect in signaling also contributes to diabetic cardiomyopathy in type 2 diabetic patients.