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Published as doi: 10.1096/fj.07-8130com.
 (The FASEB Journal. 2008;22:84-92.)
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(The FASEB Journal. 2008;22:84-92.)
© 2008 FASEB

Unmasking different mechanical and energetic roles for the small heat shock proteins CryAB and HSPB2 using genetically modified mouse hearts

Ilka Pinz*,1, Jeffrey Robbins{dagger}, Namakkal S. Rajasekaran{ddagger}, Ivor J. Benjamin{ddagger},2 and Joanne S. Ingwall*,2

* NMR Laboratory for Physiological Chemistry, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA;

{dagger} Molecular Cardiovascular Biology, Children’s Hospital, Cincinnati, Ohio, USA; and

{ddagger} Division of Cardiology, University of Utah Health Sciences Center, Salt Lake City, Utah, USA

2 Correspondence: J.S.I., Laboratory for Physiological Chemistry, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Ave, Rm. #247, Boston, MA 02115, USA. E-mail: jingwall{at}rics.bwh.harvard.edu; or I.J.B., Center for Cardiovascular Translational Biomedicine, Division of Cardiology, University of Utah Health Sciences Center, 30 North 1900 East, Salt Lake City, UT 84132, USA. E-mail: ivor.benjamin{at}hsc.utah.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
CryAB and HSPB2 are small heat shock proteins constitutively expressed in the heart. CryAB protects cytoskeletal organization and intermediate filament assembly; the functions of HSPB2 are unknown. The promoters of CryAB and HSPB2 share regulatory elements, making identifying their separate functions difficult. Here, using a genetic approach, we report distinct roles for these sHSPs, with CryAB protecting mechanical properties and HSPB2 protecting energy reserve. Isolated hearts of wild type mice (WT), mice lacking both sHSPs (DKO), WT mice overexpressing mouse CryAB protein (mCryABTg), and mice with no HSPB2 made by crossing DKO with mCryABTg (DKO/mCryABTg) were stressed with either ischemia/reperfusion or inotropic stimulation. Contractile performance and energetics were measured using 31P NMR spectroscopy. Ischemia/reperfusion caused severe diastolic dysfunction in DKO hearts. Recovery of [ATP] and [PCr] during reperfusion was impaired only in DKO/mCryABTg. During inotropic stimulation, DKO/mCryABTg showed blunted systolic and diastolic function and revealed massive energy wasting on acute stress: |{Delta}G~ATP| decreased in DKO by 6.4 ± 0.7 and in DKO/mCryABTg by 5.5 ± 0.8 kJ/mol compared with only ~3.3 kJ/mol in WT and mCryABTg. Thus, CryAB and HSPB2 proteins play nonredundant roles in the heart, CryAB in structural remodeling and HSPB2 in maintaining energetic balance.—Pinz, I., Robbins, J., Rajasekaran, N. S., Benjamin, I. J., Ingwall, J. S. Unmasking different mechanical and energetic roles for the small heat shock proteins CryAB and HSPB2 using genetically modified mouse hearts.


Key Words: transgenesis • cardiac function


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
SMALL HEAT SHOCK PROTEINS (sHSPs) are abundantly and constitutively expressed in all muscle tissues. Two members of the sHSP family, {alpha}B-Crystallin/HSPB5 (CryAB) and HSPB2, have high expression levels in the heart (1 , 2) . CryAB and HSPB2 share the highly conserved {alpha}-crystallin domain, and their promoters have shared regulatory elements, suggesting common functional roles. However, their differing intracellular distribution and interactions with other sHSPs suggest that they have distinct intracellular functions.

CryAB forms multimeric cytosolic complexes with other sHSPs (3) but not with HSPB2 or HSPB3 (4) . During stress such as that induced by heat shock, ischemia, and exercise, CryAB translocates from the cytosol to the cytoskeleton, where it binds to titin, actin, and desmin, preventing stress-induced protein aggregation and myocardial damage (5 6 7 8 9 10) . Overexpressing CryAB in mouse hearts protects myocytes from ischemic damage and also improves contractile performance post ischemia (11) . The importance of CryAB in the heart was shown when a CryAB gene mutation, which resulted in diminished chaperone activity, was linked to the development of desmin-related cardiomyopathy (DRM) (3) .

HSPB2 is the most divergent member of the sHSP family with only 30% sequence identity to all other mammalian sHSPs (12) . Little information is available about the function of HSPB2 in any tissue type in part because the promoters for CryAB and HSPB2 contain shared regulatory elements that control their coordinate expression (12 , 13) . In skeletal muscle, specific binding partners for HSPB2 are HSPB3 and myotonic dystrophy protein kinase (DMPK). In unstressed skeletal muscle, HSPB2 and HSPB3 form large oligomeric complexes in the cytosol and both the complexes and HSPB2 itself are localized on mitochondria and the neuromuscular junction (4) . During stress, the HSPB2/HSPB3 oligomeric complexes dissolve and HSPB2’s localization to mitochondria increases (9) , leading to increased cell survival. Whether the binding of HSPB2 to the outer mitochondrial membrane leads to improved energetics remains to be defined.

Three recent studies have addressed the requirements of HSPB2 and CryAB expression in ischemic cardioprotection, but whether HSPB2 and CryAB have distinct roles in the heart remains to be determined. Morrison et al. (14) first reported that isolated hearts lacking both sHSPs (DKO) exhibit severe contractile dysfunction and increased myocardial injury in response to ischemia/reperfusion ex vivo. More recently, Golenhofen et al. (10) using isolated papillary muscles have demonstrated an earlier rise in resting tension and increased postischemic contracture occurring in DKO compared with wild-type control animals after ischemia/reperfusion. In isolated cardiac myocytes, our group has similarly shown increased susceptibility of DKO compared to wild-type controls in response to simulated ischemic conditions (15) . Taken together, these studies provided new insights about the roles of these sHSPs in ischemic cardioprotection but in no study could the specific role of either HSPB2 or CryAB be unambiguously assigned (14) .

In this study, we used genetic tools to unmask distinct roles for CryAB and HSPB2 in terms of cardiac mechanics and energetics. To distinguish the effects of CryAB and HSPB2, mice lacking HSPB2 were created by crossing CryAB overexpressing mice with mice in which the genes for both CryAB and HSPB2 were deleted. This mouse line was also tested for partial or complete rescue of the CryAB phenotype. Our results suggest that CryAB and HSPB2 have distinct and nonredundant functions in the heart, with CryAB protecting diastolic performance and HSPB2 required for normal systolic performance and normal cardiac energetics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animals
Disruption of the CryAB and HSPB2 genes was generated by homologous recombination in the mouse strain 129S6 in the laboratory of Dr. E. Wawrousek at the National Eye Institute, and first described by Brady et al. (16) . The CryAB/HSPB2 null mice (DKO) are viable with 100% survival beyond 8 months (10) . Wild-type (WT; 12–20 wk of age) and age and gender matched DKO mice were studied. The protocol for generating animals used for this study was approved by the Animal Resource Committee of the University of Texas Southwestern Medical Center (Dallas, TX, USA).

To distinguish the effects of CryAB and HSPB2, mice lacking the gene only for HSPB2 were created by crossing CryAB overexpressing (by 1.5-fold) mice (mCryABTg) with the DKO mice. As described previously, mCryABTg mice were generated in the FVB/N background (17) . A breeding colony of 129S6 DKO mice was intercrossed with FVB/N mCryAB to generate the mixed genetic background and maintained by the University of Texas Southwestern Medical Center at Dallas. Note that hearts of these mice have ~50% more CryAB than WT mouse hearts. Experimental protocols were approved by the Standing Committee on Animals of Harvard Medical Area and followed the recommendations of current NIH and American Physiological Society guidelines for the use and care of laboratory animals.

Western immunoblot analysis
Hearts of WT, DKO, and mCryABTg and DKO/mCryABTg mice were isolated and supernatants of heart homogenates (18) analyzed by SDS-PAGE. Proteins were transferred to Immobilon-P membranes at 100 V (constant voltage, 1 h) using a Mini Trans-blot electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). After preincubation, the blots were incubated with the corresponding primary and secondary antibodies, before sHSPs were visualized with an enhanced chemiluminescence detection kit. The following antibodies and reagents were used. A polyclonal antibody, which recognizes both the mouse and human proteins, was raised against residues 164–175 of human CryAB. Rabbit anti-HSPB2 (aka anti-MKBP) was kindly provided by A. Suzuki (Yokohama City University, Japan). Acrylamide/bis-acrylamide, ammonium persulfate, protein assay reagent, and protein standard markers were obtained from Bio-Rad.

Isolated perfused heart preparation
Hearts of WT, DKO, mCryABTg, and DKO/mCryABTg mice were isolated and perfused in the Langendorff mode (19) . For inotropic stimulation, dobutamine was dissolved in Krebs-Henseleit buffer (15 µM stock solution) and supplied at 2% of the coronary flow via a side arm directly above the heart (final concentration=300 nM). Except for when dobutamine was present, hearts were paced at 7 Hz. Measurement of isovolumic contractile performance has been described previously (20) .

Experimental groups and protocols
With the use of 16 WT, 12 DKO, 5 mCryABTg, and 10 DKO/mCryABTg hearts, the first protocol determined isovolumic contractile performance at baseline perfusion conditions (which are defined as the steady-state condition of the heart after recovering from the isolation procedure) followed by 16 min of normothermic no-flow ischemia followed by 50 min of reperfusion. Changes in the high-energy phosphate content were measured in a subset of hearts by 31P NMR spectroscopy. In a second protocol, 9 WT, 10 DKO, 4 mCryABTg, and 8 DKO/mCryABTg hearts were perfused with 300 nM dobutamine. After measurement of baseline function and cardiac energetics, dobutamine was given for a total of 12 min; a 31P NMR spectrum was acquired during the final 8 min. At the end of experiments, hearts were blotted dry, weighed, and stored at –80°C for subsequent measurement of total creatine content (21) .

31P NMR spectroscopy
31P NMR free induction decays (FIDs) were acquired at 161.8 MHz using a Varian Inova NMR spectrometer (Varian NMR Systems, Palo Alto, CA, USA); 104 or 200 FIDs were averaged using a 60° pulse and a recycle time of 2.5 s for an overall acquisition period of 4 or 8 min. The FIDs were Fourier transformed and after phasing, baseline correction, and a line broadening of 20 Hz, the resonance areas and chemical shifts were quantified using the Varian integration program (VNMR). Intracellular pH (pHi) was determined by comparing the chemical shift of inorganic phosphate and phosphocreatine in each spectrum to values from a standard curve. For a detailed description of the calculations of metabolite concentrations and the calculation of |{Delta}G~ATP|, see Saupe et al. (20) .

Statistical analysis
Results are mean ± SE. Comparisons were performed for pairs of hearts with the same genetic background, namely WT vs. DKO and mCryABTg vs. DKO/mCryABTg with Statview (Brainpower, Calabasas, CA, USA) using one-way repeated measures ANOVA followed by Bonferroni post test comparison of means. Mean end points were compared with Student’s t test; P < 0.05 was considered significant. Linear regressions and tests of whether slopes and intercepts differed were calculated with Prism (GraphPad, San Diego, CA); P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
General characteristics of mouse hearts
Figure 1 shows results of immunoblotting heart homogenates for CryAB and HSPB2 for the two pairs of mice studied, WT vs. DKO and mCryABTg vs. DKO/mCryABTg. DKO hearts have neither CryAB nor HSPB2. The extent of overexpression of CryAB in transgenic mouse hearts (mCryABTg) is 7-fold and in hearts made by crossing DKO and mCryABTg (DKO/mCryABTg) it is ~3.5-fold (Fig. 1A, C ). HSPB2 is absent in DKO/mCryABTg mouse hearts (Fig. 1B, D ). These two pairs of mouse hearts permit comparison of hearts with either specific deficiency of either HSPB2 or both HSPB2 and CryAB expression.


Figure 1
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  		Figure 1. General characteristics of WT, DKO, mCryABTg, and DKO/mCryABTg. A, B) Representative Western blots showing the expression levels of the mCryAB and HSPB2 (NS, loading control). C, D) Densitometric quantification of mCryAB and HSPB2 levels. Transgenic overexpression of mCryAB resulted in 7-fold higher protein levels in mCryABTg compared to WT, whereas in DKO there is no detectable CryAB. Crossing of mCryABTg and DKO resulted in 3.5-fold expression of CryAB, successfully creating a mouse with no HSBP2.

Table 1 summarizes the morphological characteristics for the two pairs of mice. A consequence of deleting both HSPB2 and CryAB is a ~15% increase in LV weight and in the heart weight to body weight ratio in DKO. LV volume was unchanged. The consequences of deleting only HSPB2 differ: mCryABTg and DKO/mCryABTg hearts have similar chamber weights but LV volume was lower in DKO/mCryABTg hearts, consistent with the development of concentric hypertrophy. The different characteristics between WT and CryABTg mice are likely caused by strain dependent differences, and therefore comparisons only between the appropriate pairs of mice were done.


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  		Table 1. Presence and absence of CryAB and HSPB2 in the four groups of mice, with body-, heart-, and chamber weights

CryAB protects diastolic contractile performance during ischemia and reperfusion
To determine whether CryAB or both CryAB and HSPB2 are required for the known protective effects of these sHSPs on cardiac function following an ischemic insult, we subjected isolated perfused WT, DKO, mCryABTg, and DKO/mCryABTg hearts to 16 min normothermic global no-flow ischemia followed by 50 min of reperfusion (Fig. 2 ). Diastolic function was assessed by measuring end-diastolic pressure (EDP) and the rate of relaxation (–dP/dt), while systolic function was assessed as systolic pressure (SP) and rate of contraction (+dP/dt).


Figure 2
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  		Figure 2. Isovolumic contractile performance at baseline, during global, no-flow ischemia, and steady-state reperfusion. A–D) Comparison of the increase in EDP (end diastolic pressure) during 16 min of ischemia. DKO hearts show the earliest increase in EDP (assessed as increase over the mean value of the first six minutes of ischemia+2SD, indicated by dashed line in graphs) after 6.4 ± 0.7 min, r2 = 0.68, compared to 9.2 ± 0.8 min, r2 = 0.42 in WT (P<0.05), whereas the overexpression of CryAB in mCryABTg (C) and DKO/mCryABTg (D) delayed the increase to 10.2 ± 0.5, r2 = 0.79 and 10.7 ± 0.3 min, r2 = 0.79, respectively (P<0.05 vs. DKO, and P<0.05 mCryABTG/DKO vs. WT). Note that the magnitude of EDP at end ischemia was independent of the presence of CryAB or HSPB2. Data were fitted using a second order polynomial (using Graph Pad Prism).

Indices of contractile performance for the two pairs of hearts, WT vs. DKO and mCryABTg vs. DKO mCryABTg, during paced (7 Hz) baseline perfusion conditions were comparable (Table 2 ). During global no-flow ischemia, all hearts cease beating within seconds. The primary functional parameters that change during ischemic stress are the time when EDP increases and the magnitude of the increase. EDP began to increase earlier and the increase was greater in hearts without either CryAB or HSPB2 (Fig. 2A, B vs. C, D ). Overexpressing mCryAB (both mCryABTg and DKO/mCryABTg hearts) delayed the rise in EDP by 3–4 min and, compared to hearts with no CryAB, attenuated the increase. Thus, the time to increase in EDP was shortest (6.4±0.4 min, P<0.05 vs. WT) in DKO hearts with no CryAB and longest (10.2±0.5 min in mCryABTG and 10.7±0.3 min in mCryABTG/DKO, P<0.05) in hearts with 1.5 times normal amounts of CryAB compared to 9.2 ± 0.8 min in WT hearts. Importantly, the time to the increase in EDP was independent of the amount of HSPB2 present.


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  		Table 2. Isovolumic contractile performance at baseline and reperfusion after zero-flow ischemia

During the initial phase of the reperfusion (first 4 min), EDP decreased only in WT hearts (EDP was 26±4 mmHg in WT compared with 57±7 in DKO, 56±6 in mCryABTg, and 52±6 mmHg in DKO/mCryABTG). During steady-state reperfusion conditions (10–50 min of reperfusion), EDP remained elevated in DKO, mCryABTg, and DKO/mCryABTg, showing persistent diastolic dysfunction, but returned to near normal values in WT hearts (15±0.3 mmHg in WT compared with 37±3 in DKO, 42±6 in mCryABTg, and 38±0.1 mmHg in DKO/mCryABTg). The recovery of another parameter of diastolic performance, the rate of relaxation –dP/dt, during steady-state conditions remained blunted only in hearts containing no CryAB (DKO; Table 2 ). Taken together, these results suggest that time to contracture during ischemia and the rate, but not extent, of recovery of diastolic function during reperfusion is dependent on the presence of CryAB expression.

Although the presence of the CryAB is important for maintaining diastolic function of the ischemic heart, the presence of HSPB2 contributed to recovery of systolic performance following ischemia. Compared to their respective WT control hearts, both types of hearts with no HSPB2 recovered less well in terms of both developed pressure (even when normalized by heart size) and rate of tension development, +dP/dt (Table 2) .

HSPB2 protects cardiac energetics during ischemia and reperfusion
In skeletal muscle under stress, HSPB2 migrates to the mitochondrial membrane (6) , suggesting that HSPB2 may play a role in regulating the increase in ATP synthesis needed to support increased ATP demand during stress. This would be of particular importance in ischemia and reperfusion in the heart, where the amount of ATP is a major determinant for cell survival. To test this hypothesis, we measured changes in high energy phosphate compound ATP and in the primary energy reserve compound phosphocreatine (PCr) during ischemia and reperfusion using 31P NMR spectroscopy of isolated beating hearts. Figure 3 A shows representative 31P NMR spectra obtained for baseline perfusion conditions, end ischemia, and during steady-state reperfusion for both pairs of hearts.


Figure 3
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  		Figure 3. Cardiac energetics and systolic contractile performance at baseline end ischemia and after reperfusion. A) Summed 31P NMR spectra from four WT, DKO, and mCryABTg and DKO/mCryABTg hearts at time points: baseline, 16 min ischemia, and 50 min reperfusion. Resonance areas from left to right are: inorganic phosphate (Pi), phosphocreatine (PCr), and gamma–, alpha-, and beta-phosphoryl group of ATP. The area under the peak is directly proportional to the amount present of Pi, PCr and ATP. Note the persistent split Pi peak in the DKO/mCryABTg. B, C) ATP and PCr concentrations at baseline (filled bars) and after 50 min of reperfusion (open bars). Hearts lacking HSPB2 (DKO and DKO/mCryABTg) recover less ATP and PCr compared to their respective controls. D) Systolic performance assessed by developed pressure shows the same pattern of blunted recovery in hearts without HSPB2 as ATP and PCr. Means ± SE. * P < 0.05, using repeated measures ANOVA.

Steady-state cardiac energetics assessed as [ATP] and [PCr] of all four groups of hearts were comparable under baseline conditions (Fig. 3A ). All hearts had high ratios of [PCr] to [ATP], low [inorganic phosphate (Pi)], and normal values for intracellular pH (~7.1). With the onset of ischemia, [PCr] fell rapidly while [Pi] increased in all groups. By the end of the ischemic period, hearts containing no HSPB2 (DKO and DKO/mCryABTg) had lost significantly more ATP than either WT or mCryABTg hearts (0.8±0.4 mM in DKO vs. 3.8±1.9 mM in WT and 0.4±0.3 mM in DKO/mCryABTg vs. 1.8±0.1 mM in mCryABTg). The rate of ATP loss was significantly faster in DKO and DKO/mCryABTg hearts compared to their controls (Fig. 4 ). During reperfusion, hearts that had no HSPB2 also showed a slower and less complete recovery of cardiac energetics. [ATP] in hearts containing no HSPB2 recovered only ~30% of their preischemic values, while the corresponding control hearts recovered 40–65% of their preischemic values (Fig. 3B ). Recovery of [PCr] in hearts containing no HSPB2 was also diminished, 55–60% recovery compared to 80–100% for their respective controls (Fig. 3C ). Figure 3D shows recovery of developed pressure to highlight the similarity of the pattern.


Figure 4
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  		Figure 4. Rate of ATP loss during ischemia. Comparison of ATP loss in the two pairs, WT and DKO (A), and mCryABTg and DKO/mCryABTg (B). Loss-of-function of HSPB2 accelerates the ATP depletion during the 19 min no-flow ischemia. Slopes of regression lines are different between WT and DKO (WT: y=–41.6±9.84, DKO: y=–61.0±10.78, P=0.035) and between mCryABTg and DKO/mCryABTg (mCryABTg: y=–50.0±11.03, DKO/mCryABTg: y=–61.0±10.78, P=0.037). Means ± SE, n = 4 in each group. Significance of the differences in slopes were determined by one-way ANOVA.

HSPB2 is required for full contractile reserve
The results from the ischemia/reperfusion experiments show that HSPB2 is required for recovery of systolic performance and cardiac energetics after the relatively long stress of 16 min of ischemia and 50 min of reperfusion. To test whether HSPB2 is also required for supplying the energy needed to support an abrupt increase in work output (referred to as contractile reserve), we challenged isolated hearts with dobutamine, a strong inotropic (increase in contractile strength) and positive chronotropic (increase in heart rate) stimulus, and measured simultaneously isovolumic contractile performance and energetics. Hearts that did not contain HSPB2 (DKO vs. WT and DKO/mCryABTg vs. mCryABTg) had less contractile reserve than their corresponding controls (see Table 3 ). Although both HSPB2-deficient hearts had higher heart rates than their corresponding controls, parameters of systolic performance (systolic pressure, developed pressure, rate-pressure product and wall stress) were all significantly reduced (see Fig. 5 for wall stress values). These results suggest that the loss of HSPB2 limits contractile reserve during acute increases in cardiac work; that is, HSPB2 is required for full capacity for increasing contractile reserve.


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  		Table 3. Isovolumic contractile performance, and cardiac energetics during baseline and inotropic stimulation with dobutamine


Figure 5
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  		Figure 5. Contractile performance and cardiac energetics during inotropic stimulation with dobutamine (300 nM). A, B) Comparison of wall stress vs. |{Delta}GATP| in the 2 pairs, WT and DKO, and mCryABTg and DKO/mCryABTg. Points on the right hand side of each graph reflect baseline levels and points on the left reflect changes during dobutamine. Hearts lacking HSPB2 achieve lower wall stress but invest between 5.5 and 6.0 kJ/mol compared to ~3 kJ/mol of their respective controls. This impressive wasting of energy is also apparent when comparing the slopes of the regressions: WT: wall stress = –13.3 x +870, DKO: wall stress = –4.9 x +339, mCryABTg: wall stress = 11.5 x +715, DKO/mCryABTg: wall stress = –5.3 x +348.

HSPB2 is required for efficient coupling of the free energy of ATP hydrolysis and work during dobutamine challenge
The free energy of ATP hydrolysis, |{Delta}G~ATP|, is the chemical driving force for all ATP-requiring reactions in the cell. Here we use this term to describe the energy state of the heart, reducing all the changes that occur during an abrupt increase in work state in [ATP] (no or small decrease), [PCr] (decrease), and [Pi] (increase) to one number. During dobutamine challenge, |{Delta}G~ATP| decreased in WT and mCryABTg by 3.3 kJ/mol, a change we typically observe in control mouse hearts under these conditions. Hearts that contain no HSPB2 exhibited much larger decreases, 6.4 and 5.5 kJ/mol for DKO and DKO/mCryABTg, respectively. This impressive decrease in the energy state of hearts lacking HSPB2 is illustrated in Fig. 5 . During dobutamine challenge DKO (top panel) and DKO/mCryABTg (bottom panel,) hearts used more free energy from ATP hydrolysis to achieve less work (here shown as wall stress) as control hearts. Thus, HSPB2, independent and distinct from CryAB, is required for efficient coupling of the free energy of ATP hydrolysis and contractile performance.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Among the 10 sHSPs in the mammalian genome (22 , 23) , seven are either selectively or ubiquitously expressed in the heart. Because the promoters of CryAB and HSPB2 contain shared regulatory elements that control their tissue-restricted expression (12 , 13) , it has not been possible to assign distinct roles to these HSPs or to demonstrate whether such bidirectional gene pairing imparts unique biological properties under stress conditions (23) . Here we use a genetic approach to provide evidence that CryAB, but not HSPB2, expression mitigates ischemic diastolic dysfunction and that HSPB2, but not CryAB, expression is required for full expression of cardiac systolic function and energetics under both nonischemic and ischemic stresses. Together, these results support our hypothesis that CryAB and HSPB2 exert biologically distinct roles and that their nonredundant functions are recruited to enable the mammalian heart to respond to two important common stresses, increased work and ischemia/reperfusion.

CryAB but not HSPB2 expression delays diastolic dysfunction during ischemia/reperfusion
Using the same physiological model and the stress of zero-flow ischemia followed by reperfusion, Morrison et al. (14) recently demonstrated reduced contractile performance and increased apoptosis and necrosis in DKO mouse hearts. However, these investigators could not distinguish whether one or both sHSPs were required for ischemic protection. Here we show that a parameter used to assess diastolic dysfunction in ischemic injury, namely EDP, increased earlier in ischemic hearts with no CryAB compared with WT hearts while the time to increased EDP was similarly and significantly delayed in hearts containing 1.5-fold more mCryAB (mCryABTg and DKO/mCryABTg hearts). Importantly, the pattern was independent of the presence or absence of HSPB2. These results show that CryAB overexpression was sufficient to blunt the severe diastolic dysfunction observed in ischemic DKO hearts containing no CryAB. These data establish the cardioprotective effects of CryAB expression on diastolic performance in a whole heart preparation.

CryAB constitutes ~0.3% of the total heart protein and is the major cytosolic chaperone in the heart (14) (24) . During ischemic conditions, CryAB translocates and tightly associates with the intermediate filament desmin and elastic protein titin contained in the I-region of cardiac fibers (8) . In addition to the roles played by collagen, passive stiffness and elastic properties of the myocardium are principally determined by the repeating modules of both Ig-like and fibronectin in titin, a 3 MDa protein spanning half the sarcomere (25) . Reversible binding of CryAB to titin not only protects the extensible N2B regions but also the highly labile Ig regions of overstretched myofibrils (26) . In experiments using single molecule force spectroscopy, CryAB increased the forces for unfolding titin by stabilizing the Ig domains (26) . Despite similar force generation in isolated papillary muscles under basal conditions, there was a significantly earlier rise in resting tension and higher postischemic tension in DKO compared with muscles from WT animals. We speculate that, in the absence of CryAB in DKO hearts, many more N2B and Ig domains of titin are irreversibly denatured and aggregated (or are rendered nonfunctional by some other mechanism), precluding elastic recoil and increasing the passive stiffness during global ischemia. Conversely, the N2B domains of titin should be protected in mCryABTg and DKO/mCryABTg hearts containing 1.5-fold CryAB overexpression. Thus, the delayed increase of EDP in mCryABTg and DKO/mCryABTg hearts and improved diastolic performance reported here support the hypothesis that CryAB expression is causally linked to protection of intermediate folding states of the titin filament.

An unexpected finding was that increased CryAB expression did not protect against the magnitude of the increase in EDP either during ischemia or reperfusion: mCryABTg and DKO mCryABTg had comparable increases in EDP as in DKO and not as WT hearts (Table 2) . One possible explanation is that the degree and duration of ischemia used was severe, obliterating any differences. Another is that there may be a strict stochiometric requirement of CryAB and HSPB2 coexpression for protection and that overexpressing CryAB disrupts this balance.

Previous studies (7 , 10) using ejecting hearts overexpressing CryAB have suggested a protective effect of CryAB on the systolic contractile performance after ischemia. However, in our study, WT hearts with normal expression levels of CryAB and mCryABTg hearts with 1.5-fold higher expression CryAB levels exhibited similar postischemic recovery of systolic function, and this was the case despite differences in genetic backgrounds. The mechanisms for these disparate findings are not immediately apparent but may include the duration of the ischemic stimulus or extent of CryAB overexpression. Nonetheless, the present study for the first time uncovers a mechanistic role for CryAB in postischemic diastolic dysfunction and, in parallel, has uncovered new information about the sHSP partner, HSPB2.

HSPB2, but not CryAB, is required for maximal recruitment of contractile reserve and efficient coupling of the free energy of ATP hydrolysis and work
Combining loss-of-function and gain-of-function approaches allowed us to assign a novel role for the HSPB2. To our knowledge, this is the first time a function for HSPB2 is described. We observed that during ischemia, hearts lacking HSPB2 have a higher rate of ATP loss and that after ischemia, hearts lacking HSPB2 recover systolic function and energetics, as assessed by [ATP] and [PCr], less well than the hearts containing constitutive amounts of HSPB2 (compare Fig. 3B-D ). Hearts lacking HSPB2 also have reduced contractile reserve on acute inotropic challenge and less efficient coupling of the free energy of ATP hydrolysis and work. Importantly, this is the case whether CryAB is present or not. By using these two genetically modified mice, one with no CryAB and one with 1.5-fold overexpression of CryAB, we eliminate the possibility that the role of HSPB2 on energetics is modulated by the amount of CryAB present. These results also suggest that CryAB and HSPB2 have nonredundant roles in the mammalian heart.

We found no differences between DKO and WT hearts in the baseline energetic profile nor in the capacities of two mitochondrial enzymes: mitochondrial creatine kinase, a channel for ATP and PCr transfer from the mitochondrial matrix and the cytosol, and citrate synthase, a control point in the Krebs cycle (data not shown). We also found no differences in the maximum capacity of glycolytic enzymes (PFK, LDH, data not shown). Based on the observations that hearts lacking HSPB2 demonstrated faster loss of ATP during no-flow ischemia and a lower |{Delta}G~ATP| caused by a sudden increase in workload, our data suggest that loss of HSPB2 also reduces the glycolytic flux of the heart required for ATP production during zero-flow ischemia and abrupt increases in work. With the use of a loss of function strategy, our results show that HSPB2 expression protects against loss of ATP and PCr following severe ischemic stress. Moreover, HSPB2 is required to elicit maximal systolic performance and for efficient coupling of ATP hydrolysis and work during the normal physiological conditions of varying work. Details of the binding of HSPB2 to the outer mitochondrial membrane and its possible role in glycolysis and on the consequences on the balance between ATP synthesis and utilization as well as the compensatory role of other sHSPs remain to be defined.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
To date, the investigations reported from three independent laboratories using different approaches studying DKO hearts are noteworthy for their remarkable concordance supporting the conclusion that CryAB and HSPB2 have nonredundant roles in the mammalian heart. Despite the immediate proximity of gene localization and bidirectional pairing for CryAB and HSPB2, each sHSP imparts different and distinct functions within the cardiomyocyte. Our results support a novel role for CryAB in protection against diastolic dysfunction, perhaps on the titin filament system during myocardial ischemia. Our results further allow us to identify a novel and distinct role for HSPB2 in the heart. HSPB2 not only plays a role protecting cardiac energetics during ischemia/reperfusion, but it also allows greater capacity for increased work during acute inotropic challenge. Our findings suggest that HSPB2 is a potential future target for genetic screening among the emerging family of myofibrillar diseases of cardiac and skeletal muscle in humans.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health RO1-HL63985 (J.S.I.), SCOR-HL52320 (J.S.I.), and RO1-HL63834 (I.J.B.) and the Christi T. Smith Foundation (I.J.B.).


   FOOTNOTES
 
1 Current address: Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Dr., Scarborough, ME 04074, USA

Received for publication February 6, 2007. Accepted for publication July 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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E. Chung and L. A. Leinwand
Rescuing Cardiac Malfunction: The Roles of the Chaperone-Like Small Heat Shock Proteins
Circ. Res., December 5, 2008; 103(12): 1351 - 1353.
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